Protein Sci, 2000 Feb, 9(2), 353 - 60 Substrate- and pH-dependent contribution of oxyanion binding site to the catalysis of prolyl oligopeptidase, a paradigm of the serine oligopeptidase family; Szeltner Z et al.; Prolyl oligopeptidase, an enzyme implicated in memory disorders, is a member of a new serine peptidase family . Crystallographic studies (Fulop et al., 1998) revealed a novel oxyanion binding site containing a tyrosine residue, Tyr473 . To study the importance of Tyr473 OH, we have produced prolyl oligopeptidase and its Tyr473Phe variant in Escherichia coli . The specificity rate constant, k(cat)/Km, for the modified enzyme decreased by a factor of 8-40 with highly specific substrates, Z-Gly-Pro-Nap, and a fluorogenic octapeptide . With these compounds, the decline in k(cat) was partly compensated for by reduction in Km, a difference from the extensively studied subtilisin . With the less specific suc-Gly-Pro-Nap, the Km value, which approximates Ks, was not significantly changed, resulting in greater diminution (approximately 500-fold) in k(cat)/Km . The second-order rate constant for the reaction with Z-Pro-prolinal, a slow tight-binding transition-state analogue inhibitor, and the Ki values for a slow substrate and two product-like inhibitors were not significantly affected by the Tyr473 OH group . The mechanism of transition-state stabilization was markedly dependent upon the nature of substrate and varied with pH as the enzyme interconverted between its two catalytically competent forms. Protein Sci, 2000 Feb, 9(2), 325 - 31 Resolution of ligand positions by site-directed tryptophan fluorescence in tear lipocalin; Gasymov OK et al.; The lipocalin superfamily of proteins functions in the binding and transport of a variety of important hydrophobic molecules . Tear lipocalin is a promiscuous lipid binding member of the family and serves as a paradigm to study the molecular determinants of ligand binding . Conserved regions in the lipocalins, such as the G strand and the F-G loop, may play an important role in ligand binding and delivery . We studied structural changes in the G strand of holo- and apo-tear lipocalin using spectroscopic methods including circular dichroism analysis and site-directed tryptophan fluorescence . Apo-tear lipocalin shows the same general structural characteristics as holo-tear lipocalin including alternating periodicity of a beta-strand, orientation of amino acid residues 105, 103, 101, and 99 facing the cavity, and progressive depth in the cavity from residues 105 to 99 . For amino acid residues facing the internal aspect of cavity, the presence of a ligand is associated with blue shifted spectra . The collisional rate constants indicate that these residues are not less exposed to solvent in holo-tear lipocalin than in apo-tear lipocalin . Rather the spectral blue shifts may be accounted for by a ligand induced rigidity in holo-TL . Amino acid residues 94 and 95 are consistent with positions in the F-G loop and show greater exposure to solvent in the holo- than the apo-proteins . These findings are consistent with the general hypothesis that the F-G loop in the holo-proteins of the lipocalin family is available for receptor interactions and delivery of ligands to specific targets . Site-directed tryptophan fluorescence was used in combination with a nitroxide spin labeled fatty acid analog to elucidate dynamic ligand interactions with specific amino acid residues . Collisional quenching constants of the nitroxide spin label provide evidence that at least three amino acids of the G strand residues interact with the ligand . Stern-Volmer plots are inconsistent with a ligand that is held in a static position in the calyx, but rather suggest that the ligand is in motion . The combination of site-directed tryptophan fluorescence with quenching by nitroxide labeled species has broad applicability in probing specific interactions in the solution structure of proteins and provides dynamic information that is not attainable by X-ray crystallography. Protein Sci, 2000 Feb, 9(2), 242 - 51 Characterization of the functional role of Asp141, Asp194, and Asp464 residues in the Mn2+-L-malate binding of pigeon liver malic enzyme; Chou WY et al.; Pigeon liver malic enzyme was inactivated and cleaved at Asp141, Asp194, and Asp464 by the Cu2+-ascorbate system in acidic environment . Site-specific mutagenesis was performed at these putative metal-binding sites . Three point mutants, D141N, D194N, and D464N; three double mutants, D(141,194)N, D(194,464)N, and D(141,464)N; and a triple mutant, D(141,194,464)N; as well as the wild-type malic enzyme (WT) were successfully cloned and expressed in Escherichia coli cells . All recombinant enzymes, except the triple mutant, were purified to apparent homogeneity by successive Q-Sepharose and adenosine-2',5'-bisphosphate-agarose columns . The mutants showed similar apparent Km,NADP values to that of the WT . The Km,Mal value was increased in the D141N and D194N mutants . The Km,Mn value, on the other hand, was increased only in the D141N mutant by 14-fold, corresponding to approximately 1.6 kcal/mol for the Asp141-Mn2+ binding energy . Substrate inhibition by L-malate was only observed in WT, D464N, and D(141,464)N . Initial velocity experiments were performed to derive the various kinetic parameters . The possible interactions between Asp141, Asp194, and Asp464 were analyzed by the double-mutation cycles and triple-mutation box . There are synergistic weakening interactions between Asp141 and Asp194 in the metal binding that impel the D(141,194)N double mutant to an overall specificity constant {k(cat)/(Kd,Mn Km,Mal Km,NADP)} at least four orders of magnitude smaller than the WT value . This difference corresponds to an increase of 6.38 kcal/mol energy barrier for the catalytic efficiency . Mutation at Asp464, on the other hand, has partial additivity on the mutations at Asp141 and Asp194 . The overall specificity constants for the double mutants D(194,464)N and D(141,464)N or the triple mutant D(141,194,464)N were decreased by only 10- to 100-fold compared to the WT . These results strongly suggest the involvement of Asp141 in the Mn2+-L-malate binding for the pigeon liver malic enzyme . The Asp194 and Asp464, which may be oxidized by nonspecific binding of Cu2+, are involved in the Mn2+-L-malate binding or catalysis indirectly by modulating the binding affinity of Asp141 with the Mn2+. Protein Sci, 2000 Feb, 9(2), 213 - 7 A novel method of affinity-purifying proteins using a bis-arsenical fluorescein; Thorn KS et al.; Genetically-encoded affinity tags constitute an important strategy for purifying proteins . Here, we have designed a novel affinity matrix based on the his-arsenical fluorescein dye FlAsH, which specifically recognizes short alpha-helical peptides containing the sequence CCXXCC (Griffin BA, Adams SR, Tsien RY, 1998, Science 281:269-272) . We find that kinesin tagged with this cysteine-containing helix binds specifically to FlAsH resin and can be eluted in a fully active form . This affinity tag has several advantages over polyhistidine, the only small affinity tag in common use . The protein obtained with this single chromatographic step from crude Escherichia coli lysates is purer than that obtained with nickel affinity chromatography of 6xHis tagged kinesin . Moreover, unlike nickel affinity chromatography, which requires high concentrations of imidazole or pH changes for elution, protein bound to the FlAsH column can be completely eluted by dithiothreitol . Because of these mild elution conditions, FlAsH affinity chromatography is ideal for recovering fully active protein and for the purification of intact protein complexes. Plant Cell, 2000 Mar, 12(3), 419 - 31 The thylakoid FtsH protease plays a role in the light-induced turnover of the photosystem II D1 protein; Lindahl M et al.; The photosystem II reaction center D1 protein is known to turn over frequently . This protein is prone to irreversible damage caused by reactive oxygen species that are formed in the light; the damaged, nonfunctional D1 protein is degraded and replaced by a new copy . However, the proteases responsible for D1 protein degradation remain unknown . In this study, we investigate the possible role of the FtsH protease, an ATP-dependent zinc metalloprotease, during this process . The primary light-induced cleavage product of the D1 protein, a 23-kD fragment, was found to be degraded in isolated thylakoids in the dark during a process dependent on ATP hydrolysis and divalent metal ions, suggesting the involvement of FtsH . Purified FtsH degraded the 23-kD D1 fragment present in isolated photosystem II core complexes, as well as that in thylakoid membranes depleted of endogenous FtsH . In this study, we definitively identify the chloroplast protease acting on the D1 protein during its light-induced turnover . Unlike previously identified membrane-bound substrates for FtsH in bacteria and mitochondria, the 23-kD D1 fragment represents a novel class of FtsH substrate-functionally assembled proteins that have undergone irreversible photooxidative damage and cleavage. J Med Chem, 2000 Mar 9, 43(5), 971 - 83 Design, synthesis, and enzymatic evaluation of multisubstrate analogue inhibitors of Escherichia coli thymidine phosphorylase; Esteban-Gamboa A et al.; A series of acyclic phosphonate derivatives of thymine has been synthesized and tested as multisubstrate analogue inhibitors of Escherichia coli thymidine phosphorylase . The compounds synthesized include 1-(phosphonoalkyl)thymines with six to nine methylenes (1-4, respectively); 1-{(Z)-4-phosphonomethoxy-2-butenyl}thymine (5) and its butyl and 2,3-cis-dihydroxybutyl derivatives (6 and 7, respectively); 1-{(Z)-(4-(phosphonomethoxy)methoxy)-2-butenyl}thymine (8) and also its butyl and 2,3-cis-dihydroxybutyl analogues (9 and 10); and 1-{((Z)-4-(phosphonomethoxy)-2-butenoxy)methyl}thymine (11) . Evaluation of these compounds against E . coli revealed significant enzymatic inhibition by 2, 3, 4, 6, and 8 at a concentration of 1000 microM, 3 and 4 being the most potent . Replacement of the thymine base in 3 by 6-amino-5-bromouracil and 7-deazaxanthine afforded compounds 12 and 13, which showed a pronounced improvement of TPase inhibition, comparable to 7-deazaxanthine . When inorganic phosphate was used as a variable substrate, compounds 12 and 13 displayed competitive kinetics with respect to phosphate, indicating a direct interaction of these compounds with the phosphate binding site . Also compounds 12 and 13 were found to be competitive inhibitors of TPase against thymidine as a variable substrate . These results are consistent with the compounds being multisubstrate analogue inhibitors of E . coli TPase, and they represent the first example of such TPase inhibitors. Biochemistry, 2000 Mar 21, 39(11), 3176 - 83 Modulation of MutS ATP hydrolysis by DNA cofactors; Bjornson KP et al.; Escherichia coli MutS protein, which is required for mismatch repair, has a slow ATPase activity that obeys Michalelis-Menten kinetics . At 37 degrees C, the steady-state turnover rate for ATP hydrolysis is 1.0 +/- 0.3 min(-1) per monomer equivalent with a K(m) of 33 +/- 6 microM . Hydrolysis is competitively inhibited by the ATP analogues AMPPNP and ATPgammaS, with K(i) values of 4 microM in both cases, and by ADP with a K(i) of 40 microM . The rate of ATP hydrolysis is stimulated 2-5-fold by short hetero- and homoduplex DNAs . The concentration of DNA cofactor that yields half-maximal stimulation is lowest for oligodeoxynucleotide duplexes that contain a mismatched base pair . Pre-steady-state chemical quench analysis has demonstrated a substoichiometric initial burst of ADP formation by free MutS that is governed by a rate constant of 78 min(-1), indicating that the rate-limiting step for the steady-state reaction occurs after hydrolysis . Prebinding of MutS to homoduplex DNA does not alter the burst kinetics or amplitude but only increases the steady-state rate . In contrast, binding of the protein to heteroduplex DNA abolishes the burst of ADP formation, indicating that the rate-limiting step now occurs before hydrolysis . Gel filtration analysis indicates that the MutS dimer assembles into higher order oligomers in a concentration-dependent manner, and that ATP binding shifts this equilibrium to favor assembly . These results, together with kinetic findings, indicate nonequivalence of subunits within a MutS oligomer with respect to ATP hydrolysis and DNA binding. Biochemistry, 2000 Mar 21, 39(11), 3169 - 75 Q-band ENDOR (electron nuclear double resonance) of the high-affinity ubisemiquinone center in cytochrome bo3 from Escherichia coli; Veselov AV et al.; Electron nuclear double resonance (ENDOR) was performed on the protein-bound, stabilized, high-affinity ubisemiquinone radical, QH*-, of bo3 quinol oxidase to determine its electronic spin distribution and to probe its interaction with its surroundings . Until this present work, such ENDOR studies of protein-stabilized ubisemiquinone centers have only been done on photosynthetic reaction centers whose function is to reduce a ubiquinol pool . In contrast, QH*- serves to oxidize a ubiquinol pool in the course of electron transfer from the ubiquinol pool to the oxygen-consuming center of terminal bo3 oxidase . As documented by large hyperfine couplings (>10 MHz) to nonexchangeable protons on the QH*- ubisemiquinone ring, we provide evidence for an electronic distribution on QH*- that is different from that of the semiquinones of reaction centers . Since the ubisemiquinone itself is physically nearly identical in both QH*- and the bacterial photosynthetic reaction centers, this electronic difference is evidently a function of the local protein environment . Interaction of QH*- with this local protein environment was explicitly shown by exchangeable deuteron ENDOR that implied hydrogen bonding to the quinone and by weak proton hyperfine couplings to the local protein matrix. Biochemistry, 2000 Mar 21, 39(11), 3156 - 68 Crystal structure of Escherichia coli malate synthase G complexed with magnesium and glyoxylate at 2.0 A resolution: mechanistic implications; Howard BR et al.; The crystal structure of selenomethionine-substituted malate synthase G, an 81 kDa monomeric enzyme from Escherichia coli has been determined by MAD phasing, model building, and crystallographic refinement to a resolution of 2.0 A . The crystallographic R factor is 0.177 for 49 242 reflections observed at the incident wavelength of 1.008 A, and the model stereochemistry is satisfactory . The basic fold of the enzyme is that of a beta8/alpha8 (TIM) barrel . The barrel is centrally located, with an N-terminal alpha-helical domain flanking one side . An inserted beta-sheet domain folds against the opposite side of the barrel, and an alpha-helical C-terminal domain forms a plug which caps the active site . Malate synthase catalyzes the condensation of glyoxylate and acetyl-coenzyme A and hydrolysis of the intermediate to yield malate and coenzyme A, requiring Mg(2+) . The structure reveals an enzyme-substrate complex with glyoxylate and Mg(2+) which coordinates the aldehyde and carboxylate functions of the substrate . Two strictly conserved residues, Asp631 and Arg338, are proposed to provide concerted acid-base chemistry for the generation of the enol(ate) intermediate of acetyl-coenzyme A, while main-chain hydrogen bonds and bound Mg(2+) polarize glyoxylate in preparation for nucleophilic attack . The catalytic strategy of malate synthase appears to be essentially the same as that of citrate synthase, with the electrophile activated for nucleophilic attack by nearby positive charges and hydrogen bonds, while concerted acid-base catalysis accomplishes the abstraction of a proton from the methyl group of acetyl-coenzyme A . An active site aspartate is, however, the only common feature of these two enzymes, and the active sites of these enzymes are produced by quite different protein folds . Interesting similarities in the overall folds and modes of substrate recognition are discussed in comparisons of malate synthase with pyruvate kinase and pyruvate phosphate dikinase. Biochemistry, 2000 Mar 21, 39(11), 3134 - 40 Thiol cross-linking of cytoplasmic loops in the lactose permease of Escherichia coli; Kwaw I et al.; The N- and C-terminal halves of lactose permease, each with a single-Cys residue in a cytoplasmic loop, were coexpressed, and cross-linking was studied in the absence or presence of ligand . Out of the 68 paired-Cys mutants in cytoplasmic loops IV/V and VIII/IX or X/XI, three pairs in loop IV/V and X/XI, (i) Arg135 --> Cys/Thr338 --> Cys, (ii) Arg134 --> Cys/Val343 --> Cys, and (iii) Arg134 --> Cys/Phe345 --> Cys, form a spontaneous disulfide bond, indicating that loops IV/V and X/XI are in close proximity . In addition, specific paired-Cys residues in loop IV/V (132-138) and loop VIII/IX (282-290) or loop X/XI (335-345) cross-link with iodine and/or the homobifunctional cross-linking agents N, N'-o-phenylenedimaleimide, N,N'-p-phenylenedimaleimide, and 1, 6-bis(maleimido)hexane . The results demonstrate that loop IV/V is close to both loop VIII/IX and loop X/XI . On the other hand, similar though less extensive cross-linking studies indicate that neither the N terminus nor loop II/III appear to be close to loops VIII/IX or X/XI . The findings suggest that the longer cytoplasmic loops are highly flexible and interact in a largely random fashion . However, although a Cys residue at position 134 in loop IV/V, for example, is able to cross-link with a Cys residue at each position in loop VIII/IX or loop X/XI, Cys residues at other positions in loop IV/V exhibit markedly different cross-linking patterns . Therefore, although the domains appear to be very flexible, the interactions are not completely random, suggesting that there are probably at least some structural constraints that limit the degree of flexibility . In addition, evidence is presented suggesting that ligand binding induces conformational alterations between loop IV/V and loop VIII/IX or X/XI. Biochemistry, 2000 Mar 21, 39(11), 2931 - 9 Topography of the surface of the Escherichia coli phosphotransferase system protein enzyme IIAglc that interacts with lactose permease; Sondej M et al.; The unphosphorylated form of enzyme IIAglc of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system inhibits transport catalyzed by lactose permease . We (Seok et al . (1997) Proc . Natl . Acad . Sci . U.S.A . 94, 13515-13519) previously characterized the area on the cytoplasmic face of lactose permease that interacts with enzyme IIAglc, using radioactive enzyme IIAglc . Subsequent studies (Sondej et al . (1999) Proc . Natl . Acad . Sci . U.S.A . 96, 3525-3530) suggested consensus binding sequences on proteins that interact with enzyme IIAglc . The present study characterizes a region on the surface of enzyme IIAglc that interfaces with lactose permease . Acetylation of lysine residues by sulfosuccinimidyl acetate treatment of enzyme IIAglc, but not lactose permease, reduced the degree of interaction between the two proteins . To localize the lysine residue(s) on enzyme IIAglc that is(are) involved in the regulatory interaction, selected lysine residues were mutagenized . Conversion of nine separate lysines to glutamic acid resulted in proteins that were still capable of phosphoryl acceptance from HPr . Except for Lys69, all the modified proteins were as effective as the wild-type enzyme IIAglc in a test for binding to lactose permease . The Lys69 mutant was also defective in phosphoryl transfer to glucose permease . To derive further information concerning the contact surface, additional selected residues in the vicinity of Lys69 were mutagenized and tested for binding to lactose permease . On the basis of these studies, a model for the region of the surface of enzyme IIAglc that interacts with lactose permease is proposed. J Mol Biol, 2000 Feb 25, 296(3), 911 - 9 The GxxxG motif: a framework for transmembrane helix-helix association; Russ WP et al.; In order to identify strong transmembrane helix packing motifs, we have selected transmembrane domains exhibiting high-affinity homo-oligomerization from a randomized sequence library based on the right-handed dimerization motif of glycophorin A . Sequences were isolated using the TOXCAT system, which measures transmembrane helix-helix association in the Escherichia coli inner membrane . Strong selection was applied to a large range of sequences ( approximately 10(7) possibilities) and resulted in the identification of sequence patterns that mediate high-affinity helix-helix association . The most frequent motif isolated, GxxxG, occurs in over 80% of the isolates . Additional correlations suggest that flanking residues act in concert with the GxxxG motif, and that size complementarity is maintained at the interface, consistent with the idea that the identified sequence patterns represent packing motifs . The convergent identification of similar sequence patterns from an analysis of the transmembrane domains in the SwissProt sequence database suggests that these packing motifs are frequently utilized in naturally occurring helical membrane proteins . J Mol Biol, 2000 Feb 25, 296(3), 757 - 68 IS911 transposition is regulated by protein-protein interactions via a leucine zipper motif; Haren L et al.; Efficient intermolecular transposition of bacterial insertion sequence IS911 involves the activities of two element-encoded proteins: the transposase, OrfAB, and a regulatory factor, OrfA . OrfA shares the majority of its amino acid sequence with the N-terminal part of OrfAB . This includes a putative helix-turn-helix and three of four heptads of a leucine zipper motif . OrfA strongly stimulates OrfAB-mediated intermolecular transposition both in vivo and in vitro . The present results support the notion that this is accomplished by direct interaction between these two proteins via the leucine zipper . We used both a genetic approach, based on gene fusions with phage lambda repressor, and a physical approach, involving co-immunoprecipitation, to show that OrfA not only undergoes oligomerisation but is capable of engaging with OrfAB to form heteromultimers, and that the leucine zipper is necessary for both types of interaction . Furthermore, mutation of the leucine zipper in OrfA inactivated its regulatory function . Previous observations demonstrated that the integrity of the leucine zipper motif was also important for OrfAB binding to the IS911 terminal inverted repeats . Here, we show, in gel shift experiments, using a derivative of OrfAB deleted for the C-terminal catalytic domain, OrfAB{1-149}, that the protein is capable of pairing two inverted repeats to generate a species resembling a "synaptic complex" . Preincubation of OrfAB{1-149} with OrfA dramatically reduced formation of this complex and favored formation of an alternative complex devoid of OrfA . Together these results suggest that OrfA exerts its regulatory effect by interacting transiently with OrfAB via the leucine zipper and modifying OrfAB binding to the inverted repeats . J Mol Biol, 2000 Feb 25, 296(3), 739 - 42 A fragment of recombinant GABA(A) receptor alpha1 subunit forming rosette-like homo-oligomers; Xue H et al.; The type A gamma-aminobutyric acid (GABA(A)) receptor plays a major role in inhibitory synaptic transmission in the central nervous system . A fragment consisting of residues Cys166 to Leu296 of the alpha1 subunit of the GABA(A) receptor was overexpressed in Escherichia coli and was found to have stable beta-rich structures . Here, results from laser scattering, gel electrophoresis and electron microscopy demonstrated that this recombinant protein formed rosette-like homo-oligomers, mainly pentamers in solution . Therefore, the fragment apparently provides a valuable model system for studying the pentameric holoreceptor assembly . Non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis of the fragment showed that disulfide bonds formed between monomers contributed to the oligomerization of the fragment . The fact that this fragment alone could form pentamers in vitro strongly suggests that amino acid residues located within the Cys166-Leu296 region of the alpha1 subunit may contribute to the oligomerization of GABA(A) receptor in vivo . J Nucl Med, 2000 Mar, 41(3), 463 - 9 Radiolabeled interleukin-8: specific scintigraphic detection of infection within a few hours; van der Laken CJ et al.; Several small receptor-binding agents have been tested for imaging of infection and inflammation . The potential of chemotactic peptides and of interleukins is promising and superior to that of conventional agents . In this study, we investigated the potential of interleukin-8 (IL-8) to image infection in rabbits . METHODS: IL-8 was labeled with 123I using the Bolton-Hunter method . Twenty-fours hours after induction of Escherichia coli abscesses in the left thigh muscle, rabbits were injected intravenously with 18.5 MBq 123I-IL-8 . Gamma camera images were obtained at 5 min and at 1, 4, and 8 h after injection . Biodistribution was determined 8 h after injection . RESULTS: 123I-IL-8 rapidly cleared from the blood . Accumulation of 123I-IL-8 in the abscess was visible as early as 1 h after injection . The highest abscess uptake was obtained 4 h after injection (2.6+/-0.2 percentage injected dose {%ID}), whereas 123I-IL-8 rapidly cleared from all other tissues . This resulted in increases in abscess-to-background ratios to 13.0+/-0.7 (8 h after injection), as determined by quantification of the images . In tissue biodistribution (8 h after injection), the abscess uptake was 0.057+/-0.011 %ID/g with abscess-to-contralateral muscle ratios of 114.7+/-23.0 . The radioiodination method clearly affected the in vivo biodistribution of IL-8 because IL-8 iodinated using the lodo-Gen method cleared significantly slower from the blood and most other organs, resulting in poor visualization of the abscess . CONCLUSION: The superior characteristics of IL-8 radioiodinated using the Bolton-Hunter method--i.e., high abscess uptake and rapid background clearance within a few hours--make IL-8 a promising agent to image infection and inflammation. Circulation, 2000 Mar 14, 101(10), 1158 - 64 Construction and functional evaluation of a single-chain antibody fusion protein with fibrin targeting and thrombin inhibition after activation by factor Xa; Peter K et al.; BACKGROUND: Recombinant technology was used to produce a new anticoagulant that is preferentially localized and active at the site of the clot . METHODS AND RESULTS: The variable regions of the heavy and light chains of a fibrin-specific antibody were amplified by polymerase chain reaction (PCR) with hybridoma cDNA . To obtain a functional single-chain antibody (scFv), a linker region consisting of (Gly(4)Ser)(3) was introduced by overlap PCR . After the scFv clones were ligated with DNA encoding the pIII protein of the M13 phage, high-affinity clones were selected by 10 rounds of panning on the Bbeta15-22 peptide of fibrin (beta-peptide) . Hirudin was genetically fused to the C-terminus of the variable region of the light chain . To release the functionally essential N-terminus of hirudin at the site of a blood clot, a factor Xa recognition site was introduced between scFv(59D8) and hirudin . The fusion protein was characterized by its size on SDS-PAGE (36 kDa), by Western blotting, by its cleavage into a 29-kDa (single chain alone) and 7-kDa (hirudin) fragment, by its binding to beta-peptide, and by thrombin inhibition after Xa cleavage . Finally, the fusion protein inhibited appositional growth of whole blood clots in vitro more efficiently than native hirudin . CONCLUSIONS: A fusion protein was constructed that binds to a fibrin-specific epitope and inhibits thrombin after its activation by factor Xa . This recombinant anticoagulant effectively inhibits appositional clot growth in vitro . Its efficient and fast production at low cost should facilitate a large-scale evaluation to determine whether an effective localized antithrombin activity can be achieved without systemic bleeding complications. J Bacteriol, 2000 Apr, 182(7), 2033 - 6 Dephosphorylation of the Escherichia coli transcriptional antiterminator BglG by the sugar sensor BglF is the reversal of its phosphorylation; Chen Q et al.; The Escherichia coli BglF protein catalyzes transport and phosphorylation of beta-glucosides . In addition, BglF is a membrane sensor which reversibly phosphorylates the transcriptional regulator BglG, depending on beta-glucoside availability . Therefore, BglF has three enzymatic activities: beta-glucoside phosphotransferase, BglG phosphorylase, and phospho-BglG (BglG-P) dephosphorylase . Cys-24 of BglF is the active site which delivers the phosphoryl group either to the sugar or to BglG . To characterize the dephosphorylase activity, we asked whether BglG-P can give the phosphoryl group back to Cys-24 of BglF . Here we provide evidence which is consistent with the interpretation that Cys-24-P is an intermediate in the BglG-P dephosphorylation reaction . Hence, the dephosphorylation reaction catalyzed by BglF proceeds via reversal of the phosphorylation reaction. J Bacteriol, 2000 Apr, 182(7), 1995 - 2000 Cooperative action of the catabolite activator protein and AraC in vitro at the araFGH promoter; Johnson CM et al.; Full activation of transcription of the araFGH promoter, p(FGH), requires both the catabolite activator protein (CAP) and AraC protein . At p(FGH), the binding site for CAP is centered at position -41.5, an essential binding site for AraC is centered at position -79.5, and a second, nonessential binding site is centered at position -154.5 . In this work, we used the minimal promoter region required for in vivo activation of p(FGH) to examine the roles of CAP and AraC in stimulating formation of open complexes at p(FGH) . Migration retardation assays of open complexes showed that RNA polymerase binds exceptionally tightly to the AraC-CAP-p(FGH) complex and that the order of addition of proteins to the initiating complex is important . Similar assays with RNA polymerase containing truncated alpha subunits suggest that AraC interacts with the C-terminal domain of the alpha subunit . Finally, AraC protein also acts to prevent the improper binding of RNA polymerase at a pseudo promoter near the true p(FGH) promoter. J Bacteriol, 2000 Apr, 182(7), 1969 - 77 Regulation of rRNA transcription is remarkably robust: FIS compensates for altered nucleoside triphosphate sensing by mutant RNA polymerases at Escherichia coli rrn P1 promoters; Bartlett MS et al.; We recently identified Escherichia coli RNA polymerase (RNAP) mutants (RNAP beta' Delta215-220 and beta RH454) that form extremely unstable complexes with rRNA P1 (rrn P1) core promoters . The mutant RNAPs reduce transcription and alter growth rate-dependent regulation of rrn P1 core promoters, because the mutant RNAPs require higher concentrations of the initiating nucleoside triphosphate (NTP) for efficient transcription from these promoters than are present in vivo . Nevertheless, the mutants grow almost as well as wild-type cells, suggesting that rRNA synthesis is not greatly perturbed . We report here that the rrn transcription factor FIS activates the mutant RNAPs more strongly than wild-type RNAP, thereby compensating for the altered properties of the mutant RNAPs . FIS activates the mutant RNAPs, at least in part, by reducing the apparent K(ATP) for the initiating NTP . This and other results suggest that FIS affects a step in transcription initiation after closed-complex formation in addition to its stimulatory effect on initial RNAP binding . FIS and NTP levels increase with growth rate, suggesting that changing FIS concentrations, in conjunction with changing NTP concentrations, are responsible for growth rate-dependent regulation of rrn P1 transcription in the mutant strains . These results provide a dramatic demonstration of the interplay between regulatory mechanisms in rRNA transcription. J Bacteriol, 2000 Apr, 182(7), 1964 - 8 Repair of DNA lesions induced by hydrogen peroxide in the presence of iron chelators in Escherichia coli: participation of endonuclease IV and Fpg; Galhardo RS et al.; In Escherichia coli, the repair of lethal DNA damage induced by H(2)O(2) requires exonuclease III, the xthA gene product . Here, we report that both endonuclease IV (the nfo gene product) and exonuclease III can mediate the repair of lesions induced by H(2)O(2) under low-iron conditions . Neither the xthA nor the nfo mutants was sensitive to H(2)O(2) in the presence of iron chelators, while the xthA nfo double mutant was significantly sensitive to this treatment, suggesting that both exonuclease III and endonuclease IV can mediate the repair of DNA lesions formed under such conditions . Sedimentation studies in alkaline sucrose gradients also demonstrated that both xthA and nfo mutants, but not the xthA nfo double mutant, can carry out complete repair of DNA strand breaks and alkali-labile bonds generated by H(2)O(2) under low-iron conditions . We also found indications that the formation of substrates for exonuclease III and endonuclease IV is mediated by the Fpg DNA glycosylase, as suggested by experiments in which the fpg mutation increased the level of cell survival, as well as repair of DNA strand breaks, in an AP endonuclease-null background. J Bacteriol, 2000 Apr, 182(7), 1812 - 8 Reinvestigation of the proteolytic activity of neocarzinostatin; Heyd B et al.; Neocarzinostatin (NCS) is the most studied member of a family of chromoproteins secreted by a range of actinomycetes species . It has been proposed that in addition to their antitumoral activity related to the bound chromophores, this group of related proteins could be a secreted proteases superfamily . With the aim of dissecting the molecular basis of the proteolytic activity of NCS, an expression system allowing efficient expression of apo-NCS in Escherichia coli was constructed . The recombinant protein was properly folded and functional . Its histone-specific proteolytic activity was similar to the activity described for the natural protein . Further analyses unambiguously demonstrated that the proteolytic activity could be physically separated from NCS . This activity is therefore due not to NCS itself but to minor contaminating proteases, the nature of which differed in the recombinant and natural NCS preparations . The histone degradation test commonly used to monitor proteolytic activity is extremely sensitive and may easily generate false-positive results . These results strongly suggest that the possible proteolytic activity of the proteins of this family should be critically reconsidered. J Bacteriol, 2000 Apr, 182(7), 1788 - 93 Flavodoxin mutants of Escherichia coli K-12; Gaudu P et al.; The flavodoxins are flavin mononucleotide-containing electron transferases . Flavodoxin I has been presumed to be the only flavodoxin of Escherichia coli, and its gene, fldA, is known to belong to the soxRS (superoxide response) oxidative stress regulon . An insertion mutation of fldA was constructed and was lethal under both aerobic and anaerobic conditions; only cells that also had an intact (fldA(+)) allele could carry it . A second flavodoxin, flavodoxin II, was postulated, based on the sequence of its gene, fldB . Unlike the fldA mutant, an fldB insertion mutant is a viable prototroph in the presence or absence of oxygen . A high-copy-number fldB(+) plasmid did not complement the fldA mutation . Therefore, there must be a vital function for which FldB cannot substitute for flavodoxin I . An fldB-lacZ fusion was not induced by H(2)O(2) and is therefore not a member of the oxyR regulon . However, it displayed a soxS-dependent induction by paraquat (methyl viologen), and the fldB gene is preceded by two overlapping regions that resemble known soxS binding sites . The fldB insertion mutant did not have an increased sensitivity to the effects of paraquat on either cellular viability or the expression of a soxS-lacZ fusion . Therefore, fldB is a new member of the soxRS (superoxide response) regulon, a group of genes that is induced primarily by univalent oxidants and redox cycling compounds . However, the reactions in which flavodoxin II participates and its role during oxidative stress are unknown. J Inorg Biochem, 2000 Jan 15, 78(1), 83 - 7 The reduction of the Rieske iron-sulfur cluster in naphthalene dioxygenase by X-rays; Karlsson A et al.; Naphthalene 1,2 dioxygenase (NDO) displays characteristic UV-Vis spectra depending on the oxidation state of the Rieske center . Investigations on crystals of NDO grown for X-ray diffraction experiments showed spectra characteristic of the oxidized form . Crystals reduced in an anaerobic glovebox using sodium-dithionite showed a characteristic reduced spectrum . Spectra of crystals (cooled to 100 K) after being exposed to X-rays for data collection showed spectra corresponding to a reduced Rieske iron center, demonstrating the ability of X-rays to change the oxidation state of the Rieske iron-sulfur cluster in NDO. Bone Marrow Transplant, 2000 Mar, 25(5), 571 - 3 Constrictive pericarditis post allogeneic bone marrow transplant for Philadelphia-positive acute lymphoblastic leukaemia; Cavet J et al.; We describe two cases of severe constrictive pericarditis arising after allogeneic BMT conditioning involving total body irradiation and melphalan to treat Philadelphia-chromosome positive ALL . Both patients required pericardectomy, resulting in marked improvement in ventricular filling . However, a degree of right-sided cardiac failure persisted in both patients secondary to restrictive cardiomyopathy . Constrictive pericarditis has not been previously described after BMT, but has been observed following thoracic radiotherapy for malignancy, usually involving a substantially higher radiation dose . Pericardial constriction and restrictive cardiomyopathy should be considered as causes of breathlessness and/or oedema occurring late after BMT . Bone Marrow Transplantation (2000) 25, 571-573. Biochemistry (Mosc), 2000 Feb, 65(2), 237 - 43 Influence of nucleic acids and polysaccharides on phosphotransferase activity of preparations of secretory immunoglobulin A from human milk; Kit YY et al.; The influence of nucleic acids (DNA, tRNA), synthetic oligonucleotides, and polysaccharides (lipopolysaccharides from Escherichia coli, heparin) on protein kinase and lipid kinase activities of preparations of human secretory immunoglobulin A (sIgA) has been studied . The preparations of sIgA were isolated from human milk by chromatography on the column with Protein A-Sepharose and DEAE-sorbent (sIgA1), by affinity chromatography of sIgA1 on DNA-cellulose (sIgA2), and by gel-filtration of sIgA1 in buffer containing 5% dioxane (sIgA3) . Two 32P-labeled products with high and low electrophoretic mobility in polyacrylamide gel containing SDS were found after incubation of sIgA1 and sIgA2 with {gamma-32P}ATP . The product with low electrophoretic mobility was degraded in 10% trichloroacetic acid giving a radioactive background in lanes of the polyacrylamide gel . 32P-Labeled phospholipids were found among the phosphorylation products . Soluble and immobilized DNA increase lipid kinase activity of preparations of sIgA . In this case the secretory component and H-chains of sIgA were degraded . Fractions possessing lipid kinase activity were precipitated in the presence of heparin (1 mg/ml), and lipid kinase activity was separated from sIgA by gel-filtration in buffer containing 5% dioxane . 32P-Labeled products were formed in the presence of {gamma-32P}ATP as well as {32P}ortho-phosphoric acid . The influence of heparin and synthetic deoxy- and ribooligonucleotides on casein kinase activity of sIgA3 was studied . It was observed that deoxyribooligonucleotides in micromolar concentrations increased the rate of casein phosphorylation in the presence of sIgA3 and {gamma-32P}ATP . It has been proposed that catalytically active sIgA have an affinity to DNA (anti-DNA sIgA) and can be present in human milk as a part of lipoprotein complexes. Acta Crystallogr D Biol Crystallogr, 2000 Mar, 56 ( Pt 3), 372 - 5 Arabidopsis thaliana peroxidase N: structure of a novel neutral peroxidase; Mirza O et al.; The structure of the neutral peroxidase from Arabidopsis thaliana (ATP N) has been determined to a resolution of 1.9 A and a free R value of 20.5% . ATP N has the expected characteristic fold of the class III peroxidases, with a C(alpha) r.m.s.d . of 0.82 A when compared with horseradish peroxidase C (HRP C) . HRP C is 54% identical to ATP N in sequence . When the structures of four class III plant peroxidases are superimposed, the regions with structural differences are non-randomly distributed; all are located in one half of the molecule . The architecture of the haem pocket of ATP N is very similar to that of HRP C, in agreement with the low small-molecule substrate specificity of all class III peroxidases . The structure of ATP N suggests that the pH dependence of the substrate turnover will differ from that of HRP C owing to differences in polarity of the residues in the substrate-access channel . Since there are fewer hydrogen bonds to haem C17 propionate O atoms in ATP N than in HRP C, it is suggested that ATP N will lose haem more easily than HRP C . Unlike almost all other class III plant peroxidases, ATP N has a free cysteine residue at a similar position to the suggested secondary substrate-binding site in lignin peroxidase. Acta Crystallogr D Biol Crystallogr, 2000 Mar, 56 ( Pt 3), 359 - 62 Crystallization and preliminary X-ray analysis of the catalytic domain of the adenylate cyclase GRESAG4.1 from Trypanosoma brucei; Bieger B et al.; Adenylate cyclases (ACs) are involved in signal transduction by generating the second messenger, cAMP . In Trypanosoma brucei, 3', 5'-cyclic adenosine monophosphate (cAMP) controls the life cycle of this unicellular parasite . cAMP is generated by a class of adenylate cyclases which are either constitutively (GRESAG4.1-4.3) or transiently expressed (ESAG4) during the life cycle . Unlike mammalian ACs, the trypanosomal ACs have a simple topology comprising of a large extracellular region, a transmembrane helix and a cytosolic catalytic region . Two orthorhombic crystal forms of the catalytic AC domain of GRESAG4.1 (residues Ala884-Thr1132) were generated by the hanging-drop vapour-diffusion method . X-ray diffraction data from GRESAG4.1 crystals were collected at 1.9 A resolution using synchrotron radiation . Furthermore, two heavy-metal derivative data sets were collected from crystal form A; heavy-atom sites were subsequently located in difference Patterson maps. Acta Crystallogr D Biol Crystallogr, 2000 Mar, 56 ( Pt 3), 357 - 8 Crystallization and preliminary x-ray crystallographic analysis of NAD+-dependent DNA ligase from Thermus filiformis; Lee JY et al.; A highly thermostable DNA ligase from Thermus filiformis has been crystallized at room temperature using methoxypolyethylene glycol 5000 as a precipitant . The crystal belongs to the monoclinic space group P2(1), with unit-cell parameters a = 90.63, b = 117.80, c = 98 . 65 A, beta = 115.56 degrees . Two molecules of DNA ligase are present in the asymmetric unit, giving a crystal volume per protein mass (V(m)) of 3.1 A(3) Da(-1) and a solvent content of 61% . A native data set extending to 3.0 A resolution has been collected at 100 K using synchrotron X-rays. Acta Crystallogr D Biol Crystallogr, 2000 Mar, 56 ( Pt 3), 354 - 6 Crystallization and preliminary X-ray analysis of insect antifreeze protein from the beetle Tenebrio molitor; Liou YC et al.; Hyperactive antifreeze protein from the beetle Tenebrio molitor (TmAFP) was produced in Escherichia coli and purified by gel-permeation chromatography and HPLC . An iodinated derivative was prepared by incubating the 8.5 kDa TmAFP with N-iodosuccinimide . Native and iodinated TmAFP produced two different crystal forms when crystallized using the hanging-drop vapor-diffusion technique . Native crystals were rectangular plates that diffracted to approximately 2.5 A resolution . They were monoclinic and belonged to the space group P2(1), with unit-cell dimensions a = 38.4, b = 73.4, c = 59.3 A, beta = 97.0 degrees . Crystals of iodinated TmAFP formed elongated hexagons that allowed data to be collected to approximately 1.4 A . These crystals belonged to the space group P6(1) (or P6(5)), with unit-cell dimensions a = 73.85, b = 73.85, c = 53.15 A . There were two molecules per asymmetric unit, which corresponds to V(m) = 2.46 A(3) Da(-1) and 51% solvent content . A twofold non-crystallographic symmetry was evident from self-rotation calculations. Acta Crystallogr D Biol Crystallogr, 2000 Mar, 56 ( Pt 3), 348 - 50 Structural studies of MIP synthase; Stein AJ et al.; The conversion of glucose 6-phosphate to 1-L-myo-inositol 1--phosphate (MIP) by 1-L-myo-inositol 1-phosphate synthase (MIP synthase) is the first committed and rate-limiting step in the de novo biosynthesis of inositol in all eukaryotes . The importance of inositol-containing molecules both as membrane components and as critical second messenger signal-transduction species make the function and regulation of this enzyme important for a host of biologically important cellular functions including proliferation, neurostimulation, secretion and contraction . MIP synthase has been overexpressed in Esherichia coli and purified to homogeneity by chromatographic methods . Two crystal forms of MIP synthase were obtained by the hanging-drop vapor-diffusion method . Native data sets for both crystal forms were collected in-house on a Rigaku R-AXIS IIC imaging-plate detector . Crystal form I belongs to space group C2, with unit-cell parameters a = 153.0, b = 96.6, c = 122.6 A, beta = 126.4 degrees, and diffracts to 2.5 A resolution . Crystal form II belongs to space group P2(1), with unit-cell parameters a = 94.5, b = 186.2, c = 86.5 A, beta = 110.5 degrees, and diffracts to 2.9 A resolution. Gene, 2000 Mar 7, 245(1), 75 - 9 Cloning and characterization of cDNA encoding zebrafish Danio rerio NM23-B gene; Lee JS et al.; A full-length zebrafish NM23-B cDNA was cloned and sequenced . The zebrafish NM23-B cDNA consists of 624bp with an open reading frame of 153 amino acids . NM23-B mRNA of approximately 0.7kb is present in adult zebrafish tissues . Zebrafish NM23-B his-tagged protein (17kDa) was produced in E . coli and characterized by binding and UV-cross-linking to a single-stranded telomeric repeat (TTAGGG)(6) . This is the first report to show that fish have a NM23-H2 homologue that is similar to that in humans. Gene, 2000 Mar 7, 245(1), 59 - 63 A T-extended vector using a green fluorescent protein as an indicator; Ito Y et al.; T-extended vector (T-vector) is a useful tool for cloning PCR products directly . We exploited a novel T-vector using a green fluorescent protein (GFP) as an indicator based on insertional inactivation . The brightest GFP mutant was used for easy detection even under daylight . The 100bp and 0.9kb of PCR products were cloned, and the transformant colonies with inserts were adjudged by the fluorescent green-white screening . The GFP system was more sensitive to insertional inactivation than the beta-galactosidase system at the conventional insertion sites. FEMS Microbiol Lett, 2000 Mar 15, 184(2), 231 - 5 Evidence for the transport of zinc(II) ions via the pit inorganic phosphate transport system in Escherichia coli; Beard SJ et al.; A locus involved in zinc(II) uptake in Escherichia coli K-12 was identified through the generation of a zinc(II)-resistant mutant by transposon (Tn10dCam) mutagenesis . The mutation was located within the pitA gene, which encodes the low-affinity inorganic phosphate transport system (Pit) . The pitA mutant accumulated reduced amounts of zinc(II) when exposed to 0.5-2.0 mM ZnSO(4) during growth in Luria-Bertani medium. FEMS Microbiol Lett, 2000 Mar 15, 184(2), 225 - 9 Identification of iron-regulated genes of Helicobacter pylori by a modified fur titration assay (FURTA-Hp); Fassbinder F et al.; The Escherichia coli-based Fur titration assay (FURTA), although a powerful tool for identification of genes regulated by the ferric uptake regulator (Fur), was unsuccessful for the gastric pathogen Helicobacter pylori . The FURTA was modified by construction of an E . coli indicator strain producing H . pylori Fur only . The promoter regions of the ferric citrate receptor homolog fecA2 and the riboflavin synthesis gene ribBA were both positive in the modified FURTA, but negative in the original FURTA . Transcription of fecA2 and ribBA was demonstrated to be iron-repressed in H . pylori . This type of modification should allow FURTA analysis for bacteria with Fur binding sequences poorly recognized by E . coli Fur. FEMS Microbiol Lett, 2000 Mar 15, 184(2), 165 - 71 Small heat shock proteins, IbpA and IbpB, are involved in resistances to heat and superoxide stresses in Escherichia coli; Kitagawa M et al.; To investigate the function of Escherichia coli small heat shock proteins, IbpA and IbpB, we constructed ibpA-, ibpB- and ibpAB-overexpressing strains and also an ibpAB-disrupted strain . The ibpA-, ibpB- and ibpAB-overexpressing strains were found to be resistant not only to heat but also to superoxide stress . However, the ibpAB-disrupted strain was not more sensitive to these stresses than the wild-type strain . The heat sensitivity of a rpoH amber mutant was partially suppressed by the overexpression of plac::ibpAB . These results suggest that IbpA and IbpB may be involved in the resistances to heat and oxidative stress. J Biol Chem, 2000 Mar 17, 275(11), 8213 - 9 Stoichiometry of P1 plasmid partition complexes; Bouet JY et al.; The P1 plasmid prophage is faithfully partitioned by a high affinity nucleoprotein complex assembled at the centromere-like parS site . This partition complex is composed of P1 ParB and Escherichia coli integration host factor (IHF), bound specifically to parS . We have investigated the assembly of ParB at parS and its stoichiometry of binding . Measured by gel mobility shift assays, ParB and IHF bind tightly to parS and form a specific complex, called I + B1 . We observed that as ParB concentration was increased, a second, larger complex (I + B2) formed, followed by the formation of larger complexes, indicating that additional ParB molecules joined the initial complex . Shift Western blotting experiments indicated that the I + B2 complex contained twice as much ParB as the I + B1 complex . Using mixtures of ParB and a larger polyhistidine-tagged version of ParB (His-ParB) in DNA binding assays, we determined that the initial I + B1 complex contains one dimer of ParB . Therefore, one dimer of ParB binds to its recognition sequences that span an IHF-directed bend in parS . Once this complex forms, a second dimer can join the complex, but this assembly requires much higher ParB concentrations. J Biol Chem, 2000 Mar 17, 275(11), 8196 - 205 Purification and characterization of DnaC810, a primosomal protein capable of bypassing PriA function; Xu L et al.; Escherichia coli strains lacking PriA are severely compromised in their ability to repair UV-damaged DNA and to perform homologous recombination . These phenotypes arise because of a lack of PriA-directed replication fork assembly at recombination intermediates such as D-loops . Naturally arising suppressor mutations in dnaC restore strains carrying the priA2::kan null allele to wild-type function . We have cloned one such gene, dnaC810, and overexpressed, purified, and characterized the DnaC810 protein . DnaC810 can support a PriA-independent synthesis of phiX174 complementary strand DNA . This can be attributed to its ability, unlike wild-type DnaC, to catalyze a SSB-insensitive general priming reaction with DnaB and DnaG on any SSB-coated single-stranded DNA . Gel mobility shift analysis revealed that DnaC810 could load DnaB directly to SSB-coated single-stranded DNA as well as to D loop DNA . This explains the ability of DnaC810 to bypass the requirement for PriA, PriB, PriC, and DnaT during replication fork assembly at recombination intermediates. J Biol Chem, 2000 Mar 17, 275(11), 8103 - 13 Roles of topoisomerases in maintaining steady-state DNA supercoiling in Escherichia coli; Zechiedrich EL et al.; DNA supercoiling is essential for bacterial cell survival . We demonstrated that DNA topoisomerase IV, acting in concert with topoisomerase I and gyrase, makes an important contribution to the steady-state level of supercoiling in Escherichia coli . Following inhibition of gyrase, topoisomerase IV alone relaxed plasmid DNA to a final supercoiling density (sigma) of -0.015 at an initial rate of 0.8 links min(-1) . Topoisomerase I relaxed DNA at a faster rate, 5 links min(-1), but only to a sigma of -0.05 . Inhibition of topoisomerase IV in wild-type cells increased supercoiling to approximately the same level as in a mutant lacking topoisomerase I activity (to sigma = -0.08) . The role of topoisomerase IV was revealed by two functional assays . Removal of both topoisomerase I and topoisomerase IV caused the DNA to become hyper-negatively supercoiled (sigma = -0.09), greatly stimulating transcription from the supercoiling sensitive leu-500 promoter and increasing the number of supercoils trapped by lambda integrase site-specific recombination. J Biol Chem, 2000 Mar 17, 275(11), 8097 - 102 In the human malaria parasite Plasmodium falciparum, polyamines are synthesized by a bifunctional ornithine decarboxylase, S-adenosylmethionine decarboxylase; Muller S et al.; The polyamines putrescine, spermidine, and spermine are crucial for cell differentiation and proliferation . Interference with polyamine biosynthesis by inhibition of the rate-limiting enzymes ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC) has been discussed as a potential chemotherapy of cancer and parasitic infections . Usually both enzymes are individually transcribed and highly regulated as monofunctional proteins . We have isolated a cDNA from the malaria parasite Plasmodium falciparum that encodes both proteins on a single open reading frame, with the AdoMetDC domain in the N-terminal region connected to a C-terminal ODC domain by a hinge region . The predicted molecular mass of the entire transcript is 166 kDa . The ODC/AdoMetDC coding region was subcloned into the expression vector pASK IBA3 and transformed into the AdoMetDC- and ODC-deficient Escherichia coli cell line EWH331 . The resulting recombinant protein exhibited both AdoMetDC and ODC activity and co-eluted after gel filtration on Superdex S-200 at approximately 333 kDa, which is in good agreement with the molecular mass of approximately 326 kDa determined for the native protein from isolated P . falciparum . SDS-polyacrylamide gel electrophoresis analysis of the recombinant ODC/AdoMetDC revealed a heterotetrameric structure of the active enzyme indicating processing of the AdoMetDC domain . The data presented describe the occurrence of a unique bifunctional ODC/AdoMetDC in P . falciparum, an organization which is possibly exploitable for the design of new antimalarial drugs. J Biol Chem, 2000 Mar 17, 275(11), 8044 - 50 The role of ATP binding and hydrolysis by UvrB during nucleotide excision repair; Moolenaar GF et al.; We have isolated UvrB-DNA complexes by capture of biotinylated damaged DNA substrates on streptavidin-coated magnetic beads . With this method the UvrB-DNA preincision complex remains stable even in the absence of ATP . For the binding of UvrC to the UvrB-DNA complex no cofactor is needed . The subsequent induction of 3' incision does require ATP binding by UvrB but not hydrolysis . This ATP binding induces a conformational change in the DNA, resulting in the appearance of the DNase I-hypersensitive site at the 5' side of the damage . In contrast, the 5' incision is not dependent on ATP binding because it occurs with the same efficiency with ADP . We show with competition experiments that both incision reactions are induced by the binding of the same UvrC molecule . A DNA substrate containing damage close to the 5' end of the damaged strand is specifically bound by UvrB in the absence of UvrA and ATP (Moolenaar, G . F., Monaco, V., van der Marel, G . A., van Boom, J . H., Visse, R., and Goosen, N . (2000) J . Biol . Chem . 275, 8038-8043) . To initiate the formation of an active UvrBC-DNA incision complex, however, UvrB first needs to hydrolyze ATP, and subsequently a new ATP molecule must be bound . Implications of these findings for the mechanism of the UvrA-mediated formation of the UvrB-DNA preincision complex will be discussed. J Biol Chem, 2000 Mar 17, 275(11), 8038 - 43 The effect of the DNA flanking the lesion on formation of the UvrB-DNA preincision complex . Mechanism for the UvrA-mediated loading of UvrB onto a DNA damaged site; Moolenaar GF et al.; The UvrB-DNA preincision complex plays a key role in nucleotide excision repair in Escherichia coli . To study the formation of this complex, derivatives of a DNA substrate containing a cholesterol adduct were constructed . Introduction of a single strand nick into either the top or the bottom strand at the 3' side of the adduct stabilized the UvrB-DNA complex, most likely by the release of local stress in the DNA . Removal of both DNA strands up to the 3' incision site still allowed formation of the preincision complex . Similar modifications at the 5' side of the damage, however, gave different results . The introduction of a single strand nick at the 5' incision site completely abolished the UvrA-mediated formation of the UvrB-DNA complex . Deletion of both DNA strands up to the 5' incision site also prevented the UvrA-mediated loading of UvrB onto the damaged site, but UvrB by itself could bind very efficiently . This demonstrates that the UvrB protein is capable of recognizing damage without the matchmaker function of the UvrA protein . Our results also indicate that the UvrA-mediated loading of the UvrB protein is an asymmetric process, which starts at the 5' side of the damage. J Biol Chem, 2000 Mar 17, 275(11), 7958 - 63 Mitochondrial import and processing of wild type and type III mutant isovaleryl-CoA dehydrogenase; Volchenboum SL et al.; Isovaleric acidemia is a rare inborn error of metabolism caused by a deficiency of isovaleryl-CoA dehydrogenase (IVD), a nucleus-encoded, homotetrameric, mitochondrial flavoenzyme that catalyzes the conversion of isovaleryl-CoA to 3-methylcrotonyl-CoA . We have previously identified a nucleotide deletion in the gene for IVD in fibroblasts from a patient with isovaleric acidemia leading to a shift in reading frame and premature termination of translation . The mutant IVD precursor is imported and processed to mature size, but no active enzyme is detected in mutant fibroblasts or expressed in Escherichia coli . Examination of the crystal structure of human IVD reveals that the C terminus is involved in tetramer stability . In vitro mitochondrial import experiments show that wild type IVD protein rapidly and stably forms mature homotetramer following import, whereas Type III mutant protein never forms stable oligomers . An additional series of mutant proteins with truncations and/or alterations in the C-terminal sequence implicates the C terminus of IVD in both enzyme activity and tetramer stability . Importantly, a dimeric intermediate in the folding pathway for wild type IVD has been identified in the in vitro mitochondrial import experiments, the first report of such an intermediate in the biogenesis of an acyl-CoA dehydrogenase. J Biol Chem, 2000 Mar 17, 275(11), 7779 - 86 Kinetic characterization of the ATPase cycle of the molecular chaperone Hsc66 from Escherichia coli; Silberg JJ et al.; Hsc66 from Escherichia coli is a constitutively expressed hsp70 class molecular chaperone whose activity is coupled to ATP binding and hydrolysis . To better understand the mechanism and regulation of Hsc66, we investigated the kinetics of ATP hydrolysis and the interactions of Hsc66 with nucleotides . Steady-state experiments revealed that Hsc66 has a low affinity for ATP (K(m)(ATP) = 12.7 microM) compared with other hsp70 chaperones . The kinetics of nucleotide binding were determined by analyzing changes in the Hsc66 absorbance spectrum using stopped-flow methods at 23 degrees C . ATP binding results in a rapid, biphasic increase of Hsc66 absorbance at 280 nm; this is interpreted as arising from a two-step process in which ATP binding (k(a)(ATP) = 4.2 x 10(4) M(-1) s(-1), k(d)(ATP) = 1.1 s(-1)) is followed by a slow conformational change (k(conf) = 0 . 1 s(-1)) . Under single turnover conditions, the ATP-induced transition decays exponentially with a rate (k(decay) = 0.0013 s(-1)) similar to that observed in both steady-state and single turnover ATP hydrolysis experiments (k(hyd) = 0.0014 s(-1)) . ADP binding to Hsc66 results in a monophasic transition in the absence (k(a)(ADP) = 7 x 10(5) M(-1) s(-1), k(d)(ADP) = 60 s(-1)) and presence of physiological levels of inorganic phosphate (k(a)(ADP(P(i)) = 0.28 x 10(5) M(-1) s(-1), k(d)(ADP(P(i)) = 9.1 s(-1)) . These results indicate that ATP hydrolysis is the rate-limiting step under steady-state conditions and is >10(3)-fold slower than the rate of ADP/ATP exchange . Thus, in contrast to DnaK and eukaryotic forms of hsp70 that have been characterized to date, the R if T equilibrium balance for Hsc66 is shifted in favor of the low peptide affinity T state, and regulation of the reaction cycle is expected to occur at the ATP hydrolysis step rather than at nucleotide exchange. J Biol Chem, 2000 Mar 17, 275(11), 7708 - 12 Structural studies of lacUV5-RNA polymerase interactions in vitro . Ethylation interference and missing nucleoside analysis; Noel RJ Jr et al.; Substantial effort has been made to investigate the interactions that the Escherichia coli RNA polymerase makes with promoter DNA during transcription initiation . The lacUV5 promoter has been the object of many of these studies, and to date, an incredible wealth of information exists on how RNA polymerase interacts with this promoter . We have sought to expand current knowledge by the use of two chemical interference protocols, phosphate ethylation and missing nucleoside . We have added to existing information with the identification of additional phosphates, for example, at the start site of the template strand that, when ethylated, perturb the binding of RNA polymerase . We have also discovered a number of positions, most remarkably -37 to -34 of the nontemplate strand, where nucleoside loss decreases binding . Finally, we have discovered positions of ethylation and/or nucleoside loss that can stimulate binding . In particular, missing nucleosides and phosphate ethylation near the transcription start site enhance RNA polymerase binding. J Biol Chem, 2000 Mar 17, 275(11), 7547 - 52 Identification and functional characterization of thioredoxin from Trypanosoma brucei brucei; Reckenfelderbaumer N et al.; Trypanosomes and Leishmania, the causative agents of several tropical diseases, lack the glutathione/glutathione reductase system but have trypanothione/trypanothione reductase instead . The uniqueness of this thiol metabolism and the failure to detect thioredoxin reductases in these parasites have led to the suggestion that these protozoa lack a thioredoxin system . As presented here, this is not the case . A gene encoding thioredoxin has been cloned from Trypanosoma brucei, the causative agent of African sleeping sickness . The single copy gene, which encodes a protein of 107 amino acid residues, is expressed in all developmental stages of the parasite . The deduced protein sequence is 56% identical with a putative thioredoxin revealed by the genome project of Leishmania major . The relationship to other thioredoxins is low . T . brucei thioredoxin is unusual in having a calculated pI value of 8.5 . The gene has been overexpressed in Escherichia coli . The recombinant protein is a substrate of human thioredoxin reductase with a K(m) value of 6 microM but is not reduced by trypanothione reductase . T . brucei thioredoxin catalyzes the reduction of insulin by dithioerythritol, and functions as an electron donor for T . brucei ribonucleotide reductase . The parasite protein is the first classical thioredoxin of the order Kinetoplastida characterized so far. Chem Biol, 2000 Mar, 7(3), 185 - 96 Inhibition of Escherichia coli porphobilinogen synthase using analogs of postulated intermediates; Jarret C et al.; BACKGROUND: Porphobilinogen synthase is the second enzyme involved in the biosynthesis of natural tetrapyrrolic compounds, and condenses two molecules of 5-aminolevulinic acid (ALA) through a nonsymmetrical pathway to form porphobilinogen . Each substrate is recognized individually at two different active site positions to be regioselectively introduced into the product . According to pulse-labeling experiments, the substrate forming the propionic acid sidechain of porphobilinogen is recognized first . Two different mechanisms for the first bond-forming step between the two substrates have been proposed . The first involves carbon-carbon bond formation (an aldol-type reaction) and the second carbon-nitrogen bond formation, leading to an iminium ion . RESULTS: With the help of kinetic studies, we determined the Michaelis constants for each substrate recognition site . These results explain the Michaelis-Menten behavior of substrate analog inhibitors - they act as competitive inhibitors . Under standard conditions, however, another set of inhibitors demonstrates uncompetitive, mixed, pure irreversible, slow-binding or even quasi-irreversible inhibition behavior . CONCLUSIONS: Analysis of the different classes of inhibition behavior allowed us to make a correlation between the type of inhibition and a specific site of interaction . Analyzing the inhibition behavior of analogs of postulated intermediates strongly suggests that carbon-nitrogen bond formation occurs first. Mol Phylogenet Evol, 2000 Mar, 14(3), 342 - 52 The distribution of group I introns in lichen algae suggests that lichenization facilitates intron lateral transfer; Friedl T et al.; The nuclear-encoded small subunit ribosomal DNA gene of many lichen-forming green algae in the genus Trebouxia contains a group I intron at Escherichia coli genic position 1512 . We studied the evolutionary history of the 1512 intron in Trebouxia spp . (Trebouxiophyceae) by analyzing intron and "host" cell phylogenies . The host trees were constructed by comparing internal transcribed spacer regions of rDNA . Maximum-likelihood, maximum-parsimony, and distance analyses suggest that the 1512 intron was present in the common ancestor of the green algal classes Trebouxiophyceae, Chlorophyceae, and Ulvophyceae . The 1512 intron, however, was laterally transferred at least three times among later-diverging Trebouxia spp . that form lichen partnerships . Intron secondary structure analyses are consistent with this result . Our results support the hypothesis that lichenization may facilitate 1512 group I intron lateral transfer through the close cell-to-cell contact that occurs between the lichen algal and fungal symbionts in the developing lichen thallus . Mol Cell Biol, 2000 Apr, 20(7), 2343 - 9 Modulation of DNA binding protein affinity directly affects target site demethylation; Lin IG et al.; It has recently been shown that in Xenopus, DNA demethylation at promoter regions may involve protein-DNA interactions, based on the specificity of the demethylated sites . Utilizing a stable episomal system in human cells, we recently mapped the sites and dissected the steps of demethylation at oriP sites bound by EBNA1 protein . Although it is clear that protein binding is required for demethylation of the oriP sites, it is uncertain whether this is a unique feature of the replication origin or whether it is a general phenomenon for all DNA sequences to which sequence-specific proteins are bound . In the present study, we utilize the well-defined Escherichia coli lac repressor/operator system in human cells to determine whether protein binding to methylated DNA, in a region that is neither a replication origin nor a promoter, can also lead to demethylation of the binding sites . We found that demethylation specified by protein binding is not unique to the replication origin or to the promoter . We also found that transcriptional activity does not influence demethylation of the lac operator . Isopropyl-beta-D-thiogalactopyranoside (IPTG), an inhibitor of the lac repressor, can prevent demethylation of the lac operator DNA sites and can modulate demethylation of the lac operator by affecting the binding affinity of the lac repressor . Using this system, a titration of protein binding can be done . This titration permits one to infer that protein binding site occupancy is the determinant of demethylation at DNA sites and permits a determination of how this process progresses over time. Mutat Res, 2000 Feb 16, 459(1), 81 - 7 Dominant sensitization variants of human O(6)-methylguanine-DNA-methyltransferase obtained by a mutational screen of surface residues; Brown LR et al.; A scanning mutagenesis experiment was performed on human O(6)-methylguanine methyltransferase (hMGMT), directed largely at non-conserved surface residues that have not previously been studied . Variants typically contained two or more substitutions . Two of the 16 variants characterized in detail are inactive for methyltransfer, but increase the cytotoxicity and mutagenic effects of methylating agents . This phenotype is reminiscent of a variant (C145A) that has a mutation in the methyl-accepting cysteine . C145A is inactive, but reportedly binds methylated DNA and confers sensitivity to methylating agents . The sensitization phenotype of the two new variants is more striking in strains that are wild-type for DNA repair than in strains that are deficient for repair, suggesting that these proteins inhibit functional DNA repair proteins by competitively binding to methylated DNA . Both variants have multiple substitutions in the last helix of the protein . These results suggest that the C-terminal helix is necessary for methyltransfer activity, but not for methylguanine-specific binding. Zhonghua Gan Zang Bing Za Zhi, 2000 Feb, 8(1), 33 - 4 {Transfection and expression of HCV-NS(5)B gene in Huh-7 cells}; Fang J et al.; OBJECTIVE: Hepatitis C virus (HCV) is the major cause of blood-borne non-A, non-B hepatitis . An RNA-dependent RNA polymerase is encoded by HCV non-structural protein 5B (NS(5)B) gene . A full-length HCV RNA has been transfected into human hepatoma cells, and HCV NS(5)B has been expressed in E.Coli purified . But human hepatocyte line with high expression of NS(5)B has not been established yet . METHODS: The NS(5)B gene has been transfected into Huh-7 cells by Lipofectamine . The results of transfection were confirmed by PCR and Southern blot analysis, and the level of the NS(5)B protein in Huh-7 cells was detected by Western blot analysis . RESULTS: There were the NS(5)B sequence and the expression of HCV-RNA polymerase in Huh-7 cells transfected NS(5)B plasmids . CONCLUSIONS: We have established a HCV RNA polymerase expression system in Huh-7 cells that can be used to study the mechanism of HCV replication and to test gene therapy of hepatitis C. Zhonghua Gan Zang Bing Za Zhi, 2000 Feb, 8(1), 18 - 20 {Expression and immunological reactivity of recombinant HCV-core protein}; Dai W et al.; OBJECTIVE: To express HCV-core proteins in E.coli and to develop effective HCV-core DNA-based vaccine . METHODS: The vector that expresses the highly conserved HCV core genes were constructed . The pGEX-3X HCVCore constructs contained the 1-201 ncls (1-67aa, C201), 1-402 ncls (1-134aa, C402) and 1-591ncls ( 1-197aa, C591), then expressed in E.coli cells . RESULTS: The products of HCV C201 and C402 genes were expressed as a fusion protein with glutathione-S-transferase (GST, 26kDa) whose molecular weight were 3.1 x 10(4) and 3.9 x 10(4) separately . C591 gene was not effectively expressed in E.coli . The expressed proteins were sequestered within inclusion bodies (IB) and a variety of procedures designed to minimize IB formation proved unsuccessful . The method finally adopted involved the purification of inclusion bodies followed by the solubilization, purification, and refolding of the expressed protein . The purified C402 protein was antigenically reactive with serum from chronically infected HCV patients . BALB/C mice were immunized by a subcutaneous injection of C402 protein together with Freund's complete adjuvant which produced strong anti-HCV core humoral immune responses . CONCLUSION: It is important for the study of gene vaccine to construct a certain length of HCV core gene. Biotechnol Bioeng, 2000 Apr 20, 68(2), 218 - 30 Synthesis of bioprocesses using physical properties data; Steffens MA et al.; The aim of this article is to illustrate and evaluate a synthesis procedure which has been extended to tackle bioprocesses . Physical property information is used to screen candidate units thereby reducing the size of the synthesis problem . In this way, only units which exploit large property differences between components in a stream are selected . This is important for bioprocesses because of the large number of components and wide range of unit operations which are available . The screening technique and bioprocess-unit-design methodologies have been incorporated within an implicit enumeration algorithm which was developed for chemical process synthesis and is implemented in Java programming language . An important advantage is the ability of the bioprocess synthesis software to generate a ranked list of flowsheets which may subsequently be analyzed in more detail . Two case studies are used to evaluate the bioprocess-synthesis technique . The first system involves a product which is secreted from the host organism . The second has significantly different characteristics in that the product is intracellular and forms inclusion bodies . The latter case study, in particular, is a large synthesis problem with 12 unit operations and 20 contaminant compounds . The results show that the synthesis methodology identifies a set of economically optimal flowsheets in a reasonable computational time which demonstrates its ability to deal with large synthesis problems . Using the synthesis methodology we can generate bioprocesses which are optimal in a system-wide, rather than unit-by-unit, sense . Biotechnol Bioeng, 2000 Apr 20, 68(2), 136 - 41 Production of Sm37-GAPDH, a major therapeutical target in human schistosomiasis; Argiro L et al.; Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme in the glycolytic metabolism and the production of energy . This probably explains why GAPDH was evidenced as a major therapeutical target in several parasitic diseases; either as a vaccine candidate or as a target for chemotherapeutic treatments . Schistosoma mansoni GAPDH (Sm37-GAPDH) is one of the main schistosome vaccine candidates . The production of recombinant Sm37-GAPDH is essential to evaluate the ability of this molecule to induce protective immunity in animals and possibly in humans . The cDNA encoding Sm37-GAPDH has been cloned and sequenced . In addition, five B cell (including the major B-cell epitope Sm35-5) and two T cell epitopes have been localized on the molecule . Different expression systems have been evaluated in respect with the production yield and the GAPDH enzymatic activity . Some of them have led to either a high production of insoluble material (E . coli) or to an inactive enzyme (Pischia pastoris) . The present article describes the production setting of rSm37-GAPDH using the baculovirus-insect cell system . Large amounts of soluble rSm37-GAPDH with enzymatic activity were obtained . Most sera from individuals living in an area endemic for S . mansoni recognised the rSm37 molecule and inhibited its catalytic activity . Mol Microbiol, 2000 Mar, 35(5), 1235 - 43 Rapid inactivation of the Escherichia coli Kdp K+ uptake system by high potassium concentrations; Roe AJ et al.; The Kdp K+ uptake system of Escherichia coli is induced by limitation for K+ and/or high osmolarity . In the present study, the regulation of the activity of the Kdp system has been investigated in E . coli mutants possessing only the Kdp system as the mechanism of K+ accumulation . Cells grown in the presence of low K+ (0.1-1 mM) exhibit normal growth . However, growth inhibition results from exposure of cells to moderate levels of external K+ (> 5 mM) . Measurement of the cytoplasmic pH, of K+ pools and of transport via the Kdp system demonstrates that the Kdp system is rapidly and irreversibly inhibited by moderate external K+ . Concentrations of K+ greater than 2 mM are sufficient to cause inhibition of Kdp . At pH 6, this results in rapid lowering of the capacity for pH homeostasis, but at pH 7 the intracellular pH is unaffected . Parallel analysis of the expression of the Kdp system in a Kdp+/kdpFABC-lacZ strain shows that levels of K+ that are sufficient to inhibit Kdp activity also repress expression . As a result, growth inhibition of strains solely possessing Kdp arises jointly from inhibition of Kdp activity and repression of Kdp gene expression . These data identify an important aspect of the regulation of potassium transport via the Kdp system and also provide support for a model of regulation of Kdp expression via at least two mechanisms: sensing of both turgor and external K+ concentration. Mol Microbiol, 2000 Mar, 35(5), 1202 - 10 Two types of cold sensitivity associated with the A184-->V change in the DnaA protein; Nyborg M et al.; Multicopy dnaA(Ts) strains carrying the dnaA5 or dnaA46 allele are high-temperature resistant but are cold sensitive for colony formation . The DnaA5 and DnaA46 proteins both have an A184-->V change in the ATP binding motif of the protein, but they also have one additional mutation . The mutations were separated, and it was found that a plasmid carrying exclusively the A184-->V mutation conferred a phenotype virtually identical to that of the dnaA5 plasmid . Strains carrying plasmids with either of the additional mutations behaved like a strain carrying the dnaA+ plasmid . In temperature downshifts from 42 degrees C to 30 degrees C, chromosome replication was stimulated in the multicopy dnaA46 strain . The DNA per mass ratio increased threefold, and exponential growth was maintained for more than four mass doublings . Strains carrying plasmids with the dnaA(A184-->V) or the dnaA5 gene behaved differently . The temperature downshift resulted in run out of DNA synthesis and the strains eventually ceased growth . The arrest of DNA synthesis was not due to the inability to initiate chromosome replication because marker frequency analysis showed high initiation activity after temperature downshift . However, the marker frequencies indicated that most, if not all, of the newly initiated replication forks were stalled soon after the onset of chromosome replication . Thus, it appears that the multicopy dnaA(A184-->V) strains are cold sensitive because of an inability to elongate replication at low temperature . The multicopy dnaA46 strains, on the contrary, exhibit productive initiation and normal fork movement . In this case, the cold-sensitive phenotype may be due to DNA overproduction. Mol Microbiol, 2000 Mar, 35(5), 1065 - 78 Identification of the Escherichia coli K-12 Nramp orthologue (MntH) as a selective divalent metal ion transporter; Makui H et al.; The Escherichia coli mntH (formerly yfeP) gene encodes a putative membrane protein (MntH) highly similar to members of the eukaryotic Nramp family of divalent metal ion transporters . To determine the function of E . coli MntH, a null mutant was created and MntH was overexpressed both in wild-type E . coli and in the metal-dependent mutant hflB1(Ts) . At the restrictive temperature 42 degrees C, the mntH null mutation reduces the suppression of hflB1(Ts) thermosensitivity by exogenous divalent metals . Conversely, overexpression of MntH restores growth at 42 degrees C, increases suppression of the ts phenotype by Fe(II) and Ni(II) and renders hflB1(Ts) cells hypersensitive to Mn(II) . Transport studies in intact cells show that MntH selectively facilitates uptake of 54Mn(II) and 55Fe(II) in a temperature-, time- and proton-dependent manner . Competition studies in uptake assays and growth inhibition experiments in hflB1(Ts) mutants together indicate that MntH is a divalent metal cation transporter of broad substrate specificity . The functional characteristics of MntH suggest that it corresponds to the previously described manganese transporter of E . coli . This study indicates that proton-dependent divalent metal ion uptake has been preserved in the Nramp family from bacteria to humans. Mol Microbiol, 2000 Mar, 35(5), 961 - 73 Methylcitrate synthase from Aspergillus nidulans: implications for propionate as an antifungal agent; Brock M et al.; Aspergillus nidulans was used as a model organism to investigate the fungal propionate metabolism and the mechanism of growth inhibition by propionate . The fungus is able to grow slowly on propionate as sole carbon and energy source . Propionate is oxidized to pyruvate via the methylcitrate cycle . The key enzyme methylcitrate synthase was purified and the corresponding gene mcsA, which contains two introns, was cloned, sequenced and overexpressed in A . nidulans . The derived amino acid sequence of the enzyme shows more than 50% identity to those of most eukaryotic citrate synthases, but only 14% identity to the sequence of the recently detected bacterial methylcitrate synthase from Escherichia coli . A mcsA deletion strain was unable to grow on propionate . The inhibitory growth effect of propionate on glucose medium was enhanced in this strain, which led to the assumption that trapping of the available CoA as propionyl-CoA and/or the accumulating propionyl-CoA itself interferes with other biosynthetic pathways such as fatty acid and polyketide syntheses . In the wild-type strain, however, the predominant inhibitor may be methylcitrate . Propionate (100 mM) not only impaired hyphal growth of A . nidulans but also synthesis of the green polyketide-derived pigment of the conidia, whereas in the mutant pigmentation was abolished with 20 mM propionate. Eur J Biochem, 2000 Mar, 267(6), 1858 - 68 Exploring substrate binding and discrimination in fructose1, 6-bisphosphate and tagatose 1,6-bisphosphate aldolases; Zgiby SM et al.; Fructose 1,6-bisphosphate aldolase catalyses the reversible condensation of glycerone-P and glyceraldehyde 3-phosphate into fructose 1,6-bisphosphate . A recent structure of the Escherichia coli Class II fructose 1,6-bisphosphate aldolase {Hall, D.R., Leonard, G.A., Reed, C.D., Watt, C.I., Berry, A . & Hunter, W.N . (1999) J . Mol . Biol . 287, 383-394} in the presence of the transition state analogue phosphoglycolohydroxamate delineated the roles of individual amino acids in binding glycerone-P and in the initial proton abstraction steps of the mechanism . The X-ray structure has now been used, together with sequence alignments, site-directed mutagenesis and steady-state enzyme kinetics to extend these studies to map important residues in the binding of glyceraldehyde 3-phosphate . From these studies three residues (Asn35, Ser61 and Lys325) have been identified as important in catalysis . We show that mutation of Ser61 to alanine increases the Km value for fructose 1, 6-bisphosphate 16-fold and product inhibition studies indicate that this effect is manifested most strongly in the glyceraldehyde 3-phosphate binding pocket of the active site, demonstrating that Ser61 is involved in binding glyceraldehyde 3-phosphate . In contrast a S61T mutant had no effect on catalysis emphasizing the importance of an hydroxyl group for this role . Mutation of Asn35 (N35A) resulted in an enzyme with only 1.5% of the activity of the wild-type enzyme and different partial reactions indicate that this residue effects the binding of both triose substrates . Finally, mutation of Lys325 has a greater effect on catalysis than on binding, however, given the magnitude of the effects it is likely that it plays an indirect role in maintaining other critical residues in a catalytically competent conformation . Interestingly, despite its proximity to the active site and high sequence conservation, replacement of a fourth residue, Gln59 (Q59A) had no significant effect on the function of the enzyme . In a separate study to characterize the molecular basis of aldolase specificity, the agaY-encoded tagatose 1,6-bisphosphate aldolase of E . coli was cloned, expressed and kinetically characterized . Our studies showed that the two aldolases are highly discriminating between the diastereoisomers fructose bisphosphate and tagatose bisphosphate, each enzyme preferring its cognate substrate by a factor of 300-1500-fold . This produces an overall discrimination factor of almost 5 x 105 between the two enzymes . Using the X-ray structure of the fructose 1,6-bisphosphate aldolase and multiple sequence alignments, several residues were identified, which are highly conserved and are in the vicinity of the active site . These residues might potentially be important in substrate recognition . As a consequence, nine mutations were made in attempts to switch the specificity of the fructose 1,6-bisphosphate aldolase to that of the tagatose 1,6-bisphosphate aldolase and the effect on substrate discrimination was evaluated . Surprisingly, despite making multiple changes in the active site, many of which abolished fructose 1, 6-bisphosphate aldolase activity, no switch in specificity was observed . This highlights the complexity of enzyme catalysis in this family of enzymes, and points to the need for further structural studies before we fully understand the subtleties of the shaping of the active site for complementarity to the cognate substrate. Eur J Biochem, 2000 Mar, 267(6), 1777 - 83 Localization of the catalytic activity in restrictocin molecule by deletion mutagenesis; Nayak SK et al.; Restrictocin, produced by the fungus Aspergillus restrictus, is a highly specific ribonucleolytic toxin which cleaves a single phosphodiester bond between G4325 and A4326 in the 28S rRNA . It is a nonglycosylated, single-chain, basic protein of 149 amino acids . The putative catalytic site of restrictocin includes Tyr47, His49, Glu95, Arg120 and His136 . To map the catalytic activity in the restrictocin molecule, and to study the role of N- and C-terminus in its activity, we have systematically deleted amino-acid residues from both the termini . Three N-terminal deletions removing 8, 15 and 30 amino acids, and three C-terminal deletions lacking 4, 6, and 11 amino acids were constructed . The deletion mutants were expressed in Escherichia coli, purified to homogeneity and functionally characterized . Removal of eight N-terminal or four C-terminal amino acids rendered restrictocin partially inactive, whereas any further deletions from either end resulted in the complete inactivation of the toxin . The study demonstrates that intact N- and C-termini are required for the optimum functional activity of restrictocin. Eur J Biochem, 2000 Mar, 267(6), 1732 - 42 The role of amino-acid residues in the hydrophobic patch surrounding the haem group of cytochrome f in the interaction with plastocyanin; Gong XS et al.; Soluble turnip cytochrome f has been purified from the periplasmic fraction of Escherichia coli expressing a truncated petA gene encoding the precursor protein lacking the C-terminal 33 amino-acid residues . The protein is identical {as judged by 1H-NMR spectroscopy, midpoint redox potential (+ 365 mV) and electron transfer reactions with plastocyanin} to cytochrome f purified from turnip leaves . Several residues in the hydrophobic patch surrounding the haem group have been changed by site-directed mutagenesis, and the proteins purified from E . coli . The Y1F and Q7N mutants showed only minor changes in the plastocyanin-binding constant Ka and the second-order rate constant for electron transfer to plastocyanin, whereas the Y160S mutant showed a 30% decrease in the overall rate of electron transfer caused in part by a 60% decrease in binding constant and partially compensated by an increased driving force due to a 27-mV decrease in redox potential . In contrast, the F4Y mutant showed increased rates of electron transfer which may be ascribed to an increased binding constant and a 14-mV decrease in midpoint redox potential . This indicates that subtle changes in the hydrophobic patch can influence rates of electron transfer to plastocyanin by changing the binding constants and altering the midpoint redox potential of the cytochrome haem group. Eur J Biochem, 2000 Mar, 267(6), 1723 - 31 Suramin blocks nucleotide triphosphate binding to ribosomal protein L3 from Trypanoplasma borreli; Avliyakulov NK et al.; Ribosomal protein L3 (L3) has been demonstrated to participate in formation of the peptidyltransferase center and is essential for its catalytic activity . In the present study we show that L3 is able to bind nucleotide triphosphates with high and specific affinity in vitro . L3 was serendipitously identified by screening of a genomic phage library from a primitive kinetoplastid flagellate Trypanoplasma borreli with the ATPase domain of the topoisomerase II gene as a probe . The cloned gene was overexpressed and purified as a his-tag fusion protein in E . coli . Radioligand binding experiments, using {gamma-35S}ATP, showed that L3 is able to bind ATP but also GTP and UTP with similar high affinity (IC50 50-100 nM), while it has no ATPase activity . Furthermore, we showed that L3 has more than 500-fold higher affinity for nucleotide triphosphates compared to the corresponding nucleotide monophosphates and diphosphates . Molecular genetic and biochemical analyses allowed us to localize the NTP binding domain of L3 to the N-terminal 296 residues . Suramin, a polysulfonated naphthylamine derivative of urea, known for its chemotherapeutic effects completely inhibited the binding of {gamma-35S}ATP at subclinical levels . Results obtained with surface plasmon resonance technology showed that suramin both forms weak multimolecular complexes with L3 and binds strongly to L3 in nearly stoichiometric amounts. Eur J Biochem, 2000 Mar, 267(6), 1565 - 70 Quantitative analysis of gene expression with an improved green fluorescent protein . p6; Scholz O et al.; The fast and easy in vivo detection predestines the green fluorescent protein (GFP) for its use as a reporter to quantify promoter activities . We have increased the sensitivity of GFP detection 320-fold compared to the wild-type by constructing gfp+, which contains mutations improving the folding efficiency and the fluorescence yield of GFP+ . Twelve expression levels were measured using fusions of the gfp+ and lacZ genes with the tetA promoter in Escherichia coli . The agreement of GFP+ fluorescence with beta-galactosidase activities was excellent, demonstrating that the gfp+ gene can be used to accurately quantify gene expression in vivo . However, expression of the gfp+ gene from the stronger hsp60 promoter revealed that high cellular concentrations of GFP+ caused an inner filter effect reducing the fluorescence by 50%, thus underestimating promoter activity . This effect is probably due to the higher absorbance of cells containing GFP+ . Thus promoters with activities differing by about two orders of magnitude can be correctly quantified using the gfp+ gene . Possibilities of using GFP variants beyond this range are discussed. Eur J Biochem, 2000 Mar, 267(6), 1533 - 49 New lessons in the regulation of glucose metabolism taught by the glucose 6-phosphatase system; van de Werve G et al.; The operation of glucose 6-phosphatase (EC 3.1.3.9) (Glc6Pase) stems from the interaction of at least two highly hydrophobic proteins embedded in the ER membrane, a heavily glycosylated catalytic subunit of m 36 kDa (P36) and a 46-kDa putative glucose 6-phosphate (Glc6P) translocase (P46) . Topology studies of P36 and P46 predict, respectively, nine and ten transmembrane domains with the N-terminal end of P36 oriented towards the lumen of the ER and both termini of P46 oriented towards the cytoplasm . P36 gene expression is increased by glucose, fructose 2,6-bisphosphate (Fru-2,6-P2) and free fatty acids, as well as by glucocorticoids and cyclic AMP; the latter are counteracted by insulin . P46 gene expression is affected by glucose, insulin and cyclic AMP in a manner similar to P36 . Accordingly, several response elements for glucocorticoids, cyclic AMP and insulin regulated by hepatocyte nuclear factors were found in the Glc6Pase promoter . Mutations in P36 and P46 lead to glycogen storage disease (GSD) type-1a and type-1 non a (formerly 1b and 1c), respectively . Adenovirus-mediated overexpression of P36 in hepatocytes and in vivo impairs glycogen metabolism and glycolysis and increases glucose production; P36 overexpression in INS-1 cells results in decreased glycolysis and glucose-induced insulin secretion . The nature of the interaction between P36 and P46 in controling Glc6Pase activity remains to be defined . The latter might also have functions other than Glc6P transport that are related to Glc6P metabolism. Plant Physiol, 2000 Mar, 122(3), 887 - 94 Analysis of reductant supply systems for ferredoxin-dependent sulfite reductase in photosynthetic and nonphotosynthetic organs of maize; Yonekura-Sakakibara K et al.; Sulfite reductase (SiR) catalyzes the reduction of sulfite to sulfide in chloroplasts and root plastids using ferredoxin (Fd) as an electron donor . Using purified maize (Zea mays L.) SiR and isoproteins of Fd and Fd-NADP(+) reductase (FNR), we reconstituted illuminated thylakoid membrane- and NADPH-dependent sulfite reduction systems . Fd I and L-FNR were distributed in leaves and Fd III and R-FNR in roots . The stromal concentrations of SiR and Fd I were estimated at 1.2 and 37 microM, respectively . The molar ratio of Fd III to SiR in root plastids was approximately 3:1 . Photoreduced Fd I and Fd III showed a comparable ability to donate electrons to SiR . In contrast, when being reduced with NADPH via FNRs, Fd III showed a several-fold higher activity than Fd I . Fd III and R-FNR showed the highest rate of sulfite reduction among all combinations tested . NADP(+) decreased the rate of sulfite reduction in a dose-dependent manner . These results demonstrate that the participation of Fd III and high NADPH/NADP(+) ratio are crucial for non-photosynthetic sulfite reduction . In accordance with this view, a cysteine-auxotrophic Escherichia coli mutant defective for NADPH-dependent SiR was rescued by co-expression of maize SiR with Fd III but not with Fd I. Plant Physiol, 2000 Mar, 122(3), 635 - 44 Purification of a jojoba embryo fatty acyl-coenzyme A reductase and expression of its cDNA in high erucic acid rapeseed; Metz JG et al.; The jojoba (Simmondsia chinensis) plant produces esters of long-chain alcohols and fatty acids (waxes) as a seed lipid energy reserve . This is in contrast to the triglycerides found in seeds of other plants . We purified an alcohol-forming fatty acyl-coenzyme A reductase (FAR) from developing embryos and cloned the cDNA encoding the enzyme . Expression of a cDNA in Escherichia coli confers FAR activity upon those cells and results in the accumulation of fatty alcohols . The FAR sequence shows significant homology to an Arabidopsis protein of unknown function that is essential for pollen development . When the jojoba FAR cDNA is expressed in embryos of Brassica napus, long-chain alcohols can be detected in transmethylated seed oils . Resynthesis of the gene to reduce its A plus T content resulted in increased levels of alcohol production . In addition to free alcohols, novel wax esters were detected in the transgenic seed oils . In vitro assays revealed that B . napus embryos have an endogenous fatty acyl-coenzyme A: fatty alcohol acyl-transferase activity that could account for this wax synthesis . Thus, introduction of a single cDNA into B . napus results in a redirection of a portion of seed oil synthesis from triglycerides to waxes. Am J Respir Crit Care Med, 2000 Mar, 161(3 Pt 1), 982 - 9 Endothelin receptor blockade attenuates lipopolysaccharide-induced pulmonary nitric oxide production; Fujii Y et al.; Increased nitric oxide (NO) synthesis by the inducible nitric oxide synthase (iNOS) has been shown to contribute to the development of acute lung injury and delayed hypotension in animals injected with bacterial lipopolysaccharides (LPS) . Recent evidence indicates that endothelin-1 (ET-1) is also elevated in septic humans and in animals . To assess the contribution of ETs to LPS-induced pulmonary NO production and iNOS expression, we used P1/fl, a 22 amino acid peptide, to selectively antagonize endothelin-A receptors . Anesthetized, mechanically ventilated rats were injected with either saline or LPS (E . coli endotoxin, 20 mg/kg) and studied for 5 h . Two other groups of rats were pretreated 15 min earlier with P1/fl peptide (20 microg/kg) . Unlike saline-treated rats, rats injected with LPS showed a progressive decline in arterial pressure and a significant rise in plasma ET concentration and serum nitrite-nitrate level . In the lungs, LPS injection elicited a several-fold rise in lung iNOS activity and exhaled NO concentration and increased lung wet/dry ratio significantly . Pretreatment with P1/fl peptide eliminated the decline in arterial pressure, the rise in lung wet/dry ratio, lung NOS activity, and iNOS protein expression and significantly attenuated the increase in pulmonary exhaled NO production but had no effect on plasma ET concentration . We conclude that activation of ET-A receptors by rising ET-1 concentration enhances NO production and iNOS expression in the respiratory and vascular systems and contributes to both LPS-induced hypotension and acute lung injury. Microbiol Immunol, 2000, 44(1), 1 - 7 Genetic and biochemical properties of a hemolysin (pyolysin) produced by a swine isolate of Arcanobacterium (Actinomyces) pyogenes; Ikegami M et al.; Arcanobacterium (Actinomyces) pyogenes, a causative agent of various pyogenic diseases in domestic animals, produces a hemolysin which is thought to be an important virulence factor . This hemolysin was purified from the culture supernatant of A . pyogenes swine isolate . The purified hemolysin showed a single band with a molecular mass of 56 kDa on SDS-polyacrylamide gel electrophoresis, and its isoelectric point was 9.2 . The activity of this hemolysin was not enhanced by the addition of L-cysteine or sodium thioglycolate, but it was inhibited by cholesterol . The gene encoding the hemolysin was cloned, sequenced and expressed in Escherichia coli by means of ZAP Express vector . Analysis by SDS-polyacrylamide gel electrophoresis with immunoblotting showed that the molecular weight of the hemolysin expressed in E . coli is the same as that of the hemolysin purified from A . pyogenes . Nucleotide sequence analysis revealed an open reading frame of 1,605 bp encoding a 534 amino acid protein of 57,989 Da . The nucleotide sequence of the hemolysin gene from A . pyogenes swine isolate differed only slightly (97.6% identity) from the sequence of plo gene from A . pyogenes strain BBR1 reported by Billington et al (J . Bacteriol . 179: 6100-6106, 1997) . The cysteine residue existed in the undecapeptide region of the hemolysin, which is highly conserved in thiol-activated cytolysins (cholesterol-binding cytolysins), and is replaced with alanine . Therefore, the hemolysin of A . pyogenes seems to be a novel member of the thiol-activated cytolysin family. Obstet Gynecol, 2000 Mar, 95(3), 453 - 6 Preterm delivery in mice with renal abscess; Mussalli GM et al.; OBJECTIVE: Our purpose was to develop a mouse model of renal abscess to study the effect of extrauterine infection on preterm delivery . METHODS: Escherichia coli or sterile medium was injected into the left kidney of 70 pregnant mice that had completed approximately 75% of gestation . Preterm delivery rates were recorded for various inocula . Kidney specimens were obtained and examined grossly and histologically for abscess formation . RESULTS: Thirty-one of 51 animals (60.8%) infected with 1 x 10(5)-9 x 10(6) bacteria and none of 19 uninfected animals delivered prematurely (P < .001) . Renal abscess was induced in 100% of mice receiving bacterial inoculation but in none receiving sterile medium . CONCLUSION: Kidney injection provides a reliable method for inducing renal abscess in pregnant mice . Renal abscess induces preterm delivery at a stable rate across a wide range of bacterial inocula . This model of extrauterine infection may be particularly useful in investigations of infection-induced preterm delivery. Dig Dis Sci, 2000 Feb, 45(2), 230 - 6 Effect of bile and pancreatic juice on adenoviral-mediated gene delivery: implications on the feasibility of gene delivery through ERCP; Xie X et al.; Current research in gene delivery to the liver is focused on the intravenous, intraarterial, intraportal, or intratumoral route . Another possible route for gene delivery is via the common bile duct through endoscopic retrograde cholangiopancreatography (ERCP) . Whether bile and pancreatic juice have any effect on gene delivery is not established . To evaluate the effect of bile and pancreatic juice on adenoviral-mediated gene delivery, liver and pancreatic cell lines were infected with a recombinant adenovirus expressing an E . coli beta-galactosidase gene under the control of a cytomegalovirus promoter (rAdCMVpLacZ) in the absence or presence of various concentrations of bile and pancreatic juice . The proportion of cells infected was evaluated through X-gal staining . The toxicity of bile and pancreatic juice was also evaluated through cell morphology and detachment . Bile appeared to induce significant cytotoxicity in HepG2 and Huh7 cells (50% viability with 15 min of incubation) . Neither bile nor pancreatic juice affected transgene expression . In the absence of bile/pancreatic juice, HepG2 (15-25%) and PANC-1 cells (10-18%) were less susceptible to rAdCMVpLacZ compared to Huh7 cells (75-84%, vs HepG2, P < 0.001) and BxPc-3 (82-95%, vs PANC-1, P < 0.001) at a multiplicity of infection (MOI) of 5 . Bile reduced the transduction efficiency, but 5-10% HepG2 and 5-42% of Huh7 cells were still transduced in the presence of 80% bile for up to 10 min . Adenoviral-mediated gene delivery was reduced in the presence of pancreatic juice with a low multiplicity of infection (MOI of 5), but this effect was negated with an MOI of 50 . These data provide encouragement to develop adenoviral-mediated gene delivery through ERCP. Genetica, 1999, 106(1-2), 141 - 7 Exploring structure space . A protein structure initiative; Terwilliger TC et al.; The genome projects are changing biology by providing the genetic blueprints of entire organisms . The blueprints are tantalizing but we cannot deduce everything we need to know from them, including the structures and detailed functions of proteins . In this paper we describe an approach for obtaining structural information about proteins on a genomic scale . We describe how structural and functional information might eventually be put together to form a basis for describing life at many levels . We then describe how structural information fits into this picture and classes of proteins for which structural information would be useful in a genomic context . We conclude with a proposal for an initiative to determine protein structures on a very large scale. Genetica, 1999, 106(1-2), 85 - 92 Single-chain 434 repressors with altered DNA-binding specificities . Isolation of mutant single-chain repressors by phenotypic screening of combinatorial mutant libraries; Simoncsits A et al.; Combinatorial mutant libraries of the single-chain 434 repressor were used to discover novel DNA-binding specificities . Members of the library contain one wild type domain and one mutant domain which are connected by a recombinant peptide linker . The mutant domain contains randomized amino acids in place of the DNA-contacting residues . The single-chain derivatives are expected to recognize artificial operators containing the DNA sequence of ACAA--6 base-pairs--NNNN, where ACAA is bound by the wild-type and NNNN by the mutant domain . An in vivo library screening method was used to isolate mutant DNA-binding domains which recognize the TTAA site of an asymmetric operator . Several mutants showed high affinity binding to the selection target and also strong (up to 80 fold) preference for TTAA over the wild type TTGT sequence . Some of the isolated mutants bound with very high affinities (10-50 pM) to operators containing the TTAC sequence, a close homologue of the TTAA selection target. Genetica, 1999, 106(1-2), 49 - 55 Superhelical DNA studied by solution scattering and computer models; Langowski J et al.; We present here recent results on the structure of superhelical DNA and its changes with salt concentration between 0.01 and 1.5 M NaCl . Scattering curves of two different superhelical DNAs were determined by static light scattering . The measured radii of gyration do not change significantly with salt concentration . Small-angle neutron scattering, together with calculations from a Monte Carlo model, allows to determine the superhelix diameter . Measured and simulated scattering curves agreed almost quantitatively . Experimentally we find that the diameter decreases from 16.0 +/- 0.9 nm at 10 mM to 9.0 +/- 0.7 nm at 100 mM NaCl . The superhelix diameter from the simulated conformations decreased from 18.0 +/- 1.5 nm at 10 mM to 9.4 +/- 1.5 nm at 100 mM NaCl . At higher salt concentrations up to 1.5 M NaCl, the diameter stays constant at 9 nm. Mol Cell, 2000 Jan, 5(1), 1 - 11 The activation loop of phosphatidylinositol phosphate kinases determines signaling specificity; Kunz J et al.; Phosphatidylinositol-4,5-bisphosphate plays a pivotal role in the regulation of cell proliferation and survival, cytoskeletal reorganization, and membrane trafficking . However, little is known about the temporal and spatial regulation of its synthesis . Higher eukaryotic cells have the potential to use two distinct pathways for the generation of phosphatidylinositol-4,5-bisphosphate . These pathways require two classes of phosphatidylinositol phosphate kinases, termed type I and type II PIP kinases . While highly related by sequence, these kinases localize to different subcellular compartments, phosphorylate distinct substrates, and are functionally nonredundant . Here, we show that a 20- to 25-amino acid loop spanning the catalytic site, termed the activation loop, determines both enzymatic specificity and subcellular targeting of PIP kinases . Therefore, the activation loop controls signaling specificity and PIP kinase function at multiple levels. J Biomol NMR, 1999 Nov, 15(3), 251 - 64 HYPER: a hierarchical algorithm for automatic determination of protein dihedral-angle constraints and stereospecific C beta H2 resonance assignments from NMR data; Tejero R et al.; A new computer program, HYPER, has been developed for automated analysis of protein dihedral angle values and C beta H2 stereospecific assignments from NMR data . HYPER uses a hierarchical grid-search algorithm to determine allowed values of phi, psi, and chi 1 dihedral angles and C beta H2 stereospecific assignments based on a set of NMR-derived distance and/or scalar-coupling constraints . Dihedral-angle constraints are valuable for restricting conformational space and improving convergence in three-dimensional structure calculations . HYPER computes the set of phi, psi, and chi 1 dihedral angles and C beta H2 stereospecific assignments that are consistent with up to nine intraresidue and sequential distance bounds, two pairs of relative distance bounds, thirteen homo- and heteronuclear scalar coupling bounds, and two pairs of relative scalar coupling constant bounds . The program is designed to be very flexible, and provides for simple user modification of Karplus equations and standard polypeptide geometries, allowing it to accommodate recent and future improved calibrations of Karplus curves . The C code has been optimized to execute rapidly (0.3-1.5 CPU-sec residue-1 using a 5 degrees grid) on Silicon Graphics R8000, R10000 and Intel Pentium CPUs, making it useful for interactive evaluation of inconsistent experimental constraints . The HYPER program has been tested for internal consistency and reliability using both simulated and real protein NMR data sets. Appl Microbiol Biotechnol, 2000 Feb, 53(2), 209 - 18 Characterization and cloning of an (R)-specific trans-2,3-enoylacyl-CoA hydratase from Rhodospirillum rubrum and use of this enzyme for PHA production in Escherichia coli; Reiser SE et al.; An (R)-trans-2,3-enoylacyl-CoA hydratase was purified to near-homogeneity from Rhodospirillum rubrum . Protein sequencing of enriched protein fractions allowed the construction of a degenerate oligonucleotide . The gene encoding the (R)-specific hydratase activity was cloned following three rounds of colony hybridization using the oligonucleotide, and overexpression of the gene in E . coli led to the purification of the enzyme to homogeneity . The purified enzyme used crotonyl-CoA, trans-2,3-pentenoyl-CoA, and trans-2,3-hexenoyl-CoA with approximately equal specificity as substrates in the hydration reaction . However, no activity was observed using trans-2,3-octenoyl-CoA as a substrate, but this compound did partially inhibit crotonyl-CoA hydration . Based on the nucleotide sequence, the protein has a monomeric molecular weight of 15.4 kDa and is a homotetramer in its native form as determined by gel filtration chromatography and native PAGE . The hydratase was expressed together with the PHA synthase from Thiocapsa pfennigii in E . coli strain DH5alpha . Growth of these strains on oleic acid resulted in the production of the terpolyester poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) . Appl Microbiol Biotechnol, 2000 Feb, 53(2), 201 - 8 Production characteristics of interferon-alpha using an L-arabinose promoter system in a high-cell-density culture; Lim HK et al.; Using high-cell-density culture of Escherichia coli under the control of an L-arabinose promoter (ParaB), several factors affecting the production of recombinant protein and the formation of inclusion bodies were studied . The inducer, L-arabinose, showed a maximal induction level above 10.7 mM in the final concentration . The concentration of inducer also affected the partition of interferon-alpha (IFN-alpha) into the soluble form and inclusion bodies . Induction kinetics of the rate of accumulation of IFN-alpha on the ParaB promoter showed a slower rate than those of other promoter systems, for example T7, lac or tac . These innate characteristics of ParaB enabled cells to grow continuously in spite of the metabolic burden induced by the expression of foreign protein . The duration time of induction could control the expression of both soluble and insoluble protein . The ratio of yeast extract to glycerol (N/C ratio) in feeding media significantly affected both the production level of recombinant protein and inclusion body formation . The reason for decreasing specific bioactivity during induction can be explained by the increased proportion of inclusion bodies in the total expressed IFN-alpha. Mutat Res, 2000 Feb 16, 465(1-2), 39 - 44 Mutagenicity of benzo{a}pyrene-deoxyadenosine adducts in a sequence context derived from the p53 gene; Khalili H et al.; Mutations in the human p53 tumor suppressor gene are prominently linked to sporadic cancers in breast, lung and other tissues . Recent research has shown that tobacco-associated cancer in the human lung is related to mutation of the p53 gene mediated by the carcinogen benzo{a}pyrene (BaP), and the mutations are targeted to DNA "hot spots" at specific codons . In order to gain insight into the relation between the structures of the adducts formed by BaP at these sites and their mutagenic activities, we have synthesized site-specifically modified oligo-nucleotide adducts of the active BaP diol epoxide metabolite (anti-BaPDE) . This manuscript reports on the mutagenic consequences of replication past anti-BaPDE-deoxyadenosine adducts located within a sequence context related to codon 157 in exon 5 of the p53 gene . In this sequence context, the adduct derived from the carcinogenic 7R,8S-dihydrodiol 9S,10R-epoxide was much more active as a mutagen than the adduct derived from the noncarcinogenic 7S,8R-dihydrodiol 9R,10S-epoxide and the mutation found most frequently was an A-->G transition . Since previous studies in other sequence contexts have yielded somewhat different findings, these studies further emphasize the key role played by sequence context in determining the mutational properties of carcinogen-DNA adducts. Biochim Biophys Acta, 2000 Mar 7, 1477(1-2), 157 - 67 Structure and function of the methionine aminopeptidases; Lowther WT et al.; The removal of the N-terminal methionine from proteins and peptides is dependent upon a novel class of proteases typified by the dinuclear metalloenzyme methionine aminopeptidase from Escherichia coli (eMetAP) . Substantial progress has recently been made in determining the structures of several members of this family . The identification of human MetAP as the target of putative a