J Biol Chem, 2004 Dec 24, 279(52), 54952 - 62 Epub 2004 Sep 30. Na+-dependent K+ uptake Ktr system from the cyanobacterium Synechocystis sp . PCC 6803 and its role in the early phases of cell adaptation to hyperosmotic shock; Matsuda N et al.; Transmembrane ion transport processes play a key role in the adaptation of cells to hyperosmotic conditions . Previous work has shown that the disruption of a ktrB/ntpJ-like putative Na(+)/K(+) transporter gene in the cyanobacterium Synechocystis sp . PCC 6803 confers increased Na(+) sensitivity, and inhibits HCO(3)(-) uptake . Here, we report on the mechanistic basis of this effect . Heterologous expression experiments in Escherichia coli show that three Synechocystis genes are required for K(+) transport activity . They encode an NAD(+)-binding peripheral membrane protein (ktrA; sll0493), an integral membrane protein, belonging to a superfamily of K(+) transporters (ktrB; formerly ntpJ; slr1509), and a novel type of ktr gene product, not previously found in Ktr systems (ktrE; slr1508) . In E . coli, Synechocystis KtrABE-mediated K(+) uptake occurred with a moderately high affinity (K(m) of about 60 microm), and depended on both Na(+) and a high membrane potential, but not on ATP . KtrABE neither mediated Na(+) uptake nor Na(+) efflux . In Synechocystis sp . PCC 6803, KtrB-mediated K(+) uptake required Na(+) and was inhibited by protonophore . A Delta ktrB strain was sensitive to long term hyperosmotic stress elicited by either NaCl or sorbitol . Hyperosmotic shock led initially to loss of net K(+) from the cells . The Delta ktrB cells shocked with sorbitol failed to reaccumulate K(+) up to its original level . These data indicate that in strain PCC 6803 K(+) uptake via KtrABE plays a crucial role in the early phase of cell turgor regulation after hyperosmotic shock. Prog Lipid Res, 2004 Sep, 43(5), 383 - 402 Omega-3 fatty acids in cellular membranes: a unified concept; Valentine RC et al.; The Omega-3 fatty acid DHA (docosahexaenoic acid, 22:6) and its sister molecule EPA (eicosapentaenoic acid, 20:5) are highlighted here . These highly unsaturated fatty acids are widespread in nature, especially in the marine environment, and are essential in membranes ranging from deep sea bacteria to human neurons . Studies of DHA/EPA in bacteria have led to a working model on the structural roles of these molecules and are described in this review . The main points are: (a) genomic analysis shows that genes encoding the DHA/EPA pathways are similar, supporting the idea that structural roles in bacteria might be similar, (b) biochemical analysis shows that DHA and EPA are produced in bacteria by a polyketide process distinct from the pathway of plants and animals; this allows DHA and EPA to be produced in anaerobic or oxygen-limited environments, (c) regulatory systems triggered by temperature and pressure have been identified and studied, and add to the understanding of the roles of these molecules, (d) DHA/EPA bacteria are located almost exclusively in the marine environment, raising the prospect of an important linkage between membrane processes and marine conditions, (e) physiological studies of an EPA recombinant of E . coli show that EPA phospholipids contribute essential fluidity to the bilayer and that an EPA-enriched membrane supports a respiratory lifestyle dependent on proton bioenergetics; the EPA recombinant displays other physiological properties likely attributed to high levels of EPA in the bilayer, and (f) chemical studies such as chemical dynamic modeling support the idea that DHA and presumably EPA contribute hyperfluidizing properties to the membrane . We hypothesize that DHA/EPA phospholipids contribute fluidity and other properties to the bilayer which distinguish these highly unsaturated chains from monounsaturates and polyunsaturates such as 18:2 and 18:3 . We further hypothesize that the structural properties of DHA/EPA functioning in bacteria are also harnessed by higher organisms for enhancing crucial membrane processes including photosynthesis and energy transduction. Structure (Camb), 2004 Oct, 12(10), 1901 - 7 The crystal structure of the reduced, Zn2+-bound form of the B . subtilis Hsp33 chaperone and its implications for the activation mechanism; Janda I et al.; The bacterial heat shock protein Hsp33 is a redox-regulated chaperone activated by oxidative stress . In response to oxidation, four cysteines within a Zn2+ binding C-terminal domain form two disulfide bonds with concomitant release of the metal . This leads to the formation of the biologically active Hsp33 dimer . The crystal structure of the N-terminal domain of the E . coli protein has been reported, but neither the structure of the Zn2+ binding motif nor the nature of its regulatory interaction with the rest of the protein are known . Here we report the crystal structure of the full-length B . subtilis Hsp33 in the reduced form . The structure of the N-terminal, dimerization domain is similar to that of the E . coli protein, although there is no domain swapping . The Zn2+ binding domain is clearly resolved showing the details of the tetrahedral coordination of Zn2+ by four thiolates . We propose a structure-based activation pathway for Hsp33 . Mol Microbiol, 2004 Oct, 54(1), 251 - 63 YdgT, the Hha paralogue in Escherichia coli, forms heteromeric complexes with H-NS and StpA; Paytubi S et al.; In enteric bacteria, proteins of the Hha/YmoA family play a role in the regulation of gene expression in response to environmental factors . Interaction of both Hha and YmoA with H-NS has been reported, and an Hha/H-NS complex has been shown to modulate expression in Escherichia coli of the haemolysin operon of plasmid pHly152 . In addition to the hns gene, the chromosome of E . coli and other enteric bacteria also includes the stpA gene that encodes the StpA protein, an H-NS paralogue . We report here the identification of the Hha paralogue in E . coli, the YdgT protein . As Hha paralogue, YdgT appears to fulfil some of the functions reported for StpA as H-NS paralogue: YdgT is overexpressed in hha mutants and can compensate, at least partially, some of the hha-induced phenotypes . We also demonstrate that YdgT interacts both with H-NS and with StpA . Protein cross-linking studies showed that YdgT/H-NS heteromeric complexes are generated within the bacterial cell . The StpA protein, which is subjected to Lon-mediated turnover, was less stable in the absence of Hha or YdgT . Our findings suggest that Hha, YdgT and StpA may form complexes in vivo. Mol Microbiol, 2004 Oct, 54(1), 198 - 211 RNase HI overproduction is required for efficient full-length RNA synthesis in the absence of topoisomerase I in Escherichia coli; Baaklini I et al.; It has long been known that Escherichia coli cells deprived of topoisomerase I (topA null mutants) do not grow . Because mutations reducing DNA gyrase activity and, as a consequence, negative supercoiling, occur to compensate for the loss of topA function, it has been assumed that excessive negative supercoiling is somehow involved in the growth inhibition of topA null mutants . However, how excess negative supercoiling inhibits growth is still unknown . We have previously shown that the overproduction of RNase HI, an enzyme that degrades the RNA portion of an R-loop, can partially compensate for the growth defects because of the absence of topoisomerase I . In this article, we have studied the effects of gyrase reactivation on the physiology of actively growing topA null cells . We found that growth immediately and almost completely ceases upon gyrase reactivation, unless RNase HI is overproduced . Northern blot analysis shows that the cells have a significantly reduced ability to accumulate full-length mRNAs when RNase HI is not overproduced . Interestingly, similar phenotypes, although less severe, are also seen when bacterial cells lacking RNase HI activity are grown and treated in the same way . All together, our results suggest that excess negative supercoiling promotes the formation of R-loops, which, in turn, inhibit RNA synthesis. Mol Microbiol, 2004 Oct, 54(1), 123 - 31 Positive supercoiling is generated in the presence of Escherichia coli SeqA protein; Klungsoyr HK et al.; In Escherichia coli, the SeqA protein is known as a negative regulator of chromosome replication . This protein is also suggested to have a role in chromosome organization . SeqA preferentially binds to hemi-methylated DNA and is by immunofluorescence microscopy seen as foci situated at the replication factories . Loss of SeqA leads to increased negative supercoiling of the DNA . We show that purified SeqA protein bound to fully methylated, covalently closed or nicked circular DNA generates positive supercoils in vitro in the presence of topoisomerase I or ligase respectively . This means that binding of SeqA changes either the twist or the writhe of the DNA . The ability to affect the topology of DNA suggests that SeqA may take part in the organization of the chromosome in vivo . The topology change performed by SeqA occurred also on unmethylated plasmids . It is, however, reasonable to suppose that in vivo the major part of such activity is performed on hemi-methylated DNA at the replication factories and presumably forms the basis for the characteristic SeqA foci observed by fluorescence microscopy. Mol Microbiol, 2004 Oct, 54(1), 99 - 108 Positioning of the MinE binding site on the MinD surface suggests a plausible mechanism for activation of the Escherichia coli MinD ATPase during division site selection; Ma L et al.; Division site selection in Escherichia coli requires that the MinD protein interact with itself and with MinC and MinE . MinD is a member of the NifH-ArsA-Par-MinD subgroup of ATPases . The MinE-MinD interaction results in activation of MinD ATPase activity in the presence of membrane vesicles . The sites within MinD responsible for its interaction with MinC and MinE were studied by site-directed mutagenesis and yeast two-hybrid analysis, guided by the known three-dimensional structure of MinD proteins . This provided evidence that MinC and MinE bind to overlapping sites on the MinD surface . The results also suggested that MinE and the invariant Lys11 residue in the ATPase P-loop of MinD compete for binding to a common site within the MinD structure, thereby providing a plausible structural basis for the ability of MinE to activate the ATPase activity of MinD. Mol Microbiol, 2004 Oct, 54(1), 60 - 74 Cell cycle-dependent abundance, stability and localization of FtsA and FtsQ in Caulobacter crescentus; Martin ME et al.; Coordination between cell division and DNA replication is ensured by checkpoints that act through proteins required for cell division . Following a block in DNA replication, transcription of the cell division progression genes ftsA and ftsQ is prevented in Caulobacter crescentus . One requirement for this checkpoint is that FtsA and/or FtsQ should be limiting for division in the next cell cycle . We show that the number of FtsA and FtsQ molecules fluctuates such that their concentration is low in swarmer and stalked cells, peaks in pre-divisional cells, and then dramatically decreases after cell division . Despite constitutive expression from an inducible promoter, FtsA and FtsQ levels still vary during the cell cycle, and the half-life of FtsA increases from 13 min in swarmer cells to 55 min in stalked cell types, confirming cell type-specific degradation . The post-division degradation of FtsA and FtsQ in swarmer cells reduces their concentration to 7% and 10% of their maximal level, respectively, strongly suggesting that de novo synthesis of both proteins is required for each division cycle . The localization of FtsA and FtsQ is also cell type-specific . FtsA and FtsQ are recruited to the midcell during a short period in late pre-divisional cells, consistent with the demonstrated requirement of FtsA for late stages of cell division . As previously reported for FtsZ, constitutive expression of FtsA causes cell division defects . These results indicate that the tight control of FtsA, and probably FtsQ, by cell cycle transcription, proteolysis, and localization are critical for optimal cell division and the coordination of cell division with the DNA replication cycle. Mol Microbiol, 2004 Oct, 54(1), 45 - 59 Versatility of the carboxy-terminal domain of the alpha subunit of RNA polymerase in transcriptional activation: use of the DNA contact site as a protein contact site for MarA; Dangi B et al.; The transcriptional activator, MarA, interacts with RNA polymerase (RNAP) to activate promoters of the mar regulon . Here, we identify the interacting surfaces of MarA and of the carboxy-terminal domain of the alpha subunit of RNAP (alpha-CTD) by NMR-based chemical shift mapping . Spectral changes were monitored for a MarA-DNA complex upon titration with alpha-CTD, and for alpha-CTD upon titration with MarA-DNA . The mapping results were confirmed by mutational studies and retention chromatography . A model of the ternary complex shows that alpha-CTD uses a '265-like determinant' to contact MarA at a surface distant from the DNA . This is unlike the interaction of alpha-CTD with the CRP or Fis activators where the '265 determinant' contacts DNA while another surface of the same alpha-CTD molecule contacts the activator . These results reveal a new versatility for alpha-CTD in transcriptional activation. Biotechnol Prog, 2004 Sep-Oct, 20(5), 1599 - 604 Increasing the acetyl-CoA pool in the presence of overexpressed phosphoenolpyruvate carboxylase or pyruvate carboxylase enhances succinate production in Escherichia coli; Lin H et al.; An in vivo strategy to apply the activation effect of acetyl-CoA on phosphoenolpyruvate carboxylase (PEPC) and pyruvate carboxylase (PYC) to increase succinate production in Escherichia coli was studied . This approach relies on the increased intracellular acetyl-CoA and CoA levels by overexpressing E . coli pantothenate kinase (PANK) . The results showed that coexpression of PANK and PEPC, and PANK and PYC, did improve succinate production compared to the individual expression of PEPC and PYC, respectively . The intracellular acetyl-CoA and CoA levels were also measured, and each showed a significant increase when the PANK was overexpressed . Another effect observed was a decrease in lactate production . The least amount of lactate was produced when PANK and PEPC, and PANK and PYC, were coexpressed . This result showed increased competitiveness of the succinate pathway at the phosphoenolpyruvate and pyruvate nodes for the carbon flux, as a result reducing the carbon flux toward the lactate pathway . The study also demonstrates a feasible method for metabolic engineering to modulate enzyme activity in vivo through specific activators and inhibitors. Biotechnol Prog, 2004 Sep-Oct, 20(5), 1572 - 7 A thermostable sugar-binding protein from the Archaeon Pyrococcus horikoshii as a probe for the development of a stable fluorescence biosensor for diabetic patients; Staiano M et al.; In this work is presented the first attempt to develop an innovative ultrastable protein-based biosensor for blood glucose detections . The gene of a putative thermostable sugar-binding protein has been cloned and expressed in E . coli . The recombinant protein has been purified to homogeneity by thermoprecipitation and affinity chromatography steps . The recombinant protein is a monomer with an apparent molecular weight of 55,000 as judged by gel filtration and sodium dodecyl sulfate polyacrylamide gel eletrophoresis . Circular dichroism experiments showed that the protein possesses a secondary structure content rich in alpha-helices and beta-structures and that the protein is highly stable as investigated in the range of temperature between 20 and 95 degrees C . Fluorescence spectroscopy experiments demonstrated that the recombinant protein binds glucose with a dissociation constant of about 10 mM, a concentration of sugar very close to the concentration of glucose present in the human blood . A docking simulation on the modeled structure of the protein confirms its ability to bind glucose and proposes possible modifications to improve the affinity for glucose and/or its detection . The obtained results suggest the use of the protein as a probe for a stable glucose biosensor. Biotechnol Prog, 2004 Sep-Oct, 20(5), 1512 - 7 Moderate temperatures affect Escherichia coli inactivation by high-pressure homogenization only through fluid viscosity; Diels AM et al.; The inactivation of suspensions of Escherichia coli MG1655 by high-pressure homogenization was studied over a wide range of pressures (100-300 MPa) and initial temperatures of the samples (5-50 degrees C) . Bacterial inactivation was positively correlated with the applied pressure and with the initial temperature . When samples were adjusted to different concentrations of poly(ethylene glycol) to have the same viscosity at different temperatures below 45 degrees C and then homogenized at these temperatures, no difference in inactivation was observed . These observations strongly suggest, for the first time, that the influence of temperature on bacterial inactivation by high-pressure homogenization is only through its effect on fluid viscosity . At initial temperatures > or =45 degrees C, corresponding to an outlet sample temperature >65 degrees C, the level of inactivation was higher than what would be predicted on the basis of the reduced viscosity at these temperatures, suggesting that under these conditions heat starts to contribute to cellular inactivation in addition to the mechanical effects that are predominant at lower temperatures . Second-order polynomial models were proposed to describe the impact of a high-pressure homogenization treatment of E . coli MG1655 as a function of pressure and temperature or as a function of pressure and viscosity . The pressure-viscosity inactivation model provided a better quality of fit of the experimental data and furthermore is more comprehensive and versatile than the pressure-temperature model because in addition to viscosity it implicitly incorporates temperature as a variable. Biotechnol Prog, 2004 Sep-Oct, 20(5), 1301 - 8 Role of arginine in protein refolding, solubilization, and purification; Tsumoto K et al.; Recombinant proteins are often expressed in the form of insoluble inclusion bodies in bacteria . To facilitate refolding of recombinant proteins obtained from inclusion bodies, 0.1 to 1 M arginine is customarily included in solvents used for refolding the proteins by dialysis or dilution . In addition, arginine at higher concentrations, e.g., 0.5-2 M, can be used to extract active, folded proteins from insoluble pellets obtained after lysing Escherichia coli cells . Moreover, arginine increases the yield of proteins secreted to the periplasm, enhances elution of antibodies from Protein-A columns, and stabilizes proteins during storage . All these arginine effects are apparently due to suppression of protein aggregation . Little is known, however, about the mechanism . Various effects of solvent additives on proteins have been attributed to their preferential interaction with the protein, effects on surface tension, or effects on amino acid solubility . The suppression of protein aggregation by arginine cannot be readily explained by either surface tension effects or preferential interactions . In this review we show that interactions between the guanidinium group of arginine and tryptophan side chains may be responsible for suppression of protein aggregation by arginine. World J Gastroenterol, 2004 Nov 1, 10(21), 3205 - 7 Cloning and sequence analysis of gene oipA encoding an outer membrane protein of human Helicobacter pylori; Chen DR et al.; AIM: To construct a recombinant E . coli strain that would highly express the proinflammatory outer membrane protein of human Helicobacter pylori (H pylori) . METHODS: The oipA DNA was amplified by PCR, inserted into pET-32a, and transformed into Top10 E . coli strain . This recombinant plasmid of Top10 was sent out for nucleotide sequence analysis . Finally this sequence AF479754 was compared with HP0638 and JHP0581 . RESULTS: The sequence of the aim gene was obtained . It had 924 base pairs . The identity was 95.32% against HP0638, 95.02% against JHP0581, which was higher than the identity between HP0638 and JHP0581 . CONCLUSION: Although the aim gene was obtained, but it was different from the published sequence of GenBank . It is not clear what makes this difference . Maybe it is because different strain was used or because there were some variations . So more researches are required to prove it. EMBO J, 2004 Oct 13, 23(20), 3909 - 17 Epub 2004 Sep 30. Crystal structure of human GGA1 GAT domain complexed with the GAT-binding domain of Rabaptin5; Zhu G et al.; GGA proteins coordinate the intracellular trafficking of clathrin-coated vesicles through their interaction with several other proteins . The GAT domain of GGA proteins interacts with ARF, ubiquitin, and Rabaptin5 . The GGA-Rabaptin5 interaction is believed to function in the fusion of trans-Golgi-derived vesicles to endosomes . We determined the crystal structure of a human GGA1 GAT domain fragment in complex with the Rabaptin5 GAT-binding domain . In this structure, the Rabaptin5 domain is a 90-residue-long helix . At the N-terminal end, it forms a parallel coiled-coil homodimer, which binds one GAT domain of GGA1 . In the C-terminal region, it further assembles into a four-helix bundle tetramer . The Rabaptin5-binding motif of the GGA1 GAT domain consists of a three-helix bundle . Thus, the binding between Rabaptin5 and GGA1 GAT domain is based on a helix bundle-helix bundle interaction . The current structural observation is consistent with previously reported mutagenesis data, and its biological relevance is further confirmed by new mutagenesis studies and affinity analysis . The four-helix bundle structure of Rabaptin5 suggests a functional role in tethering organelles. EMBO J, 2004 Oct 13, 23(20), 4029 - 39 Epub 2004 Sep 30. ACF1 improves the effectiveness of nucleosome mobilization by ISWI through PHD-histone contacts; Eberharter A et al.; The nucleosome remodelling ATPase ISWI resides in several distinct protein complexes whose subunit composition reflects their functional specialization . Association of ISWI with ACF1, the largest subunit of CHRAC and ACF complexes, improves the efficiency of ISWI-induced nucleosome mobilization by an order of magnitude and also modulates the reaction qualitatively . In order to understand the principle by which ACF1 improves the efficiency of ISWI, we mapped their mutual interaction requirements and generated a series of ACF complexes lacking conserved ACF1 domains . Deletion of the C-terminal PHD finger modules of ACF1 or their disruption by zinc chelation profoundly affected the nucleosome mobilization capability of associated ISWI in trans . Interactions of the PHD fingers with the central domains of core histones contribute significantly to the binding of ACF to the nucleosome substrate, suggesting a novel role for PHD modules as nucleosome interaction determinants . Connecting ACF to histones may be prerequisite for efficient conversion of ATP-dependent conformational changes of ISWI into translocation of DNA relative to the histones during nucleosome mobilization. J Biol Chem, 2004 Dec 10, 279(50), 52694 - 702 Epub 2004 Dec 10. Characterization of a new member of the flavoprotein disulfide reductase family of enzymes from Mycobacterium tuberculosis; Argyrou A et al.; The lpdA (Rv3303c) gene from Mycobacterium tuberculosis encoding a new member of the flavoprotein disulfide reductases was expressed in Escherichia coli, and the recombinant LpdA protein was purified to homogeneity . LpdA is a homotetramer and co-purifies with one molecule of tightly but noncovalently bound FAD and NADP+ per monomer . Although annotated as a probable lipoamide dehydrogenase in M . tuberculosis, LpdA cannot catalyze reduction of lipoyl substrates, because it lacks one of two cysteine residues involved in dithiol-disulfide interchange with lipoyl substrates and a His-Glu pair involved in general acid catalysis . The crystal structure of LpdA was solved by multiple isomorphous replacement with anomalous scattering, which confirmed the absence of these catalytic residues from the active site . Although LpdA cannot catalyze reduction of disulfide-bonded substrates, it catalyzes the NAD(P)H-dependent reduction of alternative electron acceptors such as 2,6-dimethyl-1,4-benzoquinone and 5-hydroxy-1,4-naphthaquinone . Significant primary deuterium kinetic isotope effects were observed with {4S-2H}NADH establishing that the enzyme promotes transfer of the C4-proS hydride of NADH . The absence of an isotope effect with {4S-2H}NADPH, the low Km value of 0.5 microm for NADPH, and the potent inhibition of the NADH-dependent reduction of 2,6-dimethyl-1,4-benzoquinone by NADP+ (Ki approximately 6 nm) and 2'-phospho-ADP-ribose (Ki approximately 800 nm), demonstrate the high affinity of LpdA for 2'-phosphorylated nucleotides and that the physiological substrate/product pair is NADPH/NADP+ rather than NADH/NAD+ . Modeling of NADP+ in the active site revealed that LpdA achieves the high specificity for NADP+ through interactions involving the 2'-phosphate of NADP+ and amino acid residues that are different from those in glutathione reductase. J Biol Chem, 2004 Dec 3, 279(49), 50949 - 55 Epub 2004 Dec 3. Use of molecular simulation for mapping conformational CYP2E1 epitopes; Vidali M et al.; The identification of the epitopes recognized by autoantibodies against cytochrome P450s (CYPs) associated with drug-induced hepatotoxicity is difficult because of their conformational nature . In the present investigation, we used a novel approach based on the analysis of the whole molecule antigenic capacity following single amino acid substitutions to identify the conformational epitopes on CYP2E1 . A molecular model of CYP2E1 was generated based on the CYP2C5 crystal structure, and potential motifs for amino acid exchanges were selected by computer simulation in the surface of alpha helices and beta sheets . Fourteen modified, apparently correctly folded CYP2E1 variants were produced in Escherichia coli and evaluated in immunoprecipitation experiments using sera with anti-CYP2E1 autoreactivity from 10 patients with halothane hepatitis and 12 patients with alcoholic liver disease . Ala substitution of Glu-248 and Lys-251 as well as of Lys-324, Lys-342, Lys-420, and Phe-421 severely decreased or abolished CYP2E1 recognition by the majority of both the halothane hepatitis and alcoholic liver disease sera, whereas the other substitutions had only minor effects . Based on the structural model, these substitutions identified two distinct epitopes on the CYP2E1 surface corresponding to the G-helix and an area formed by juxtaposition of the J' and K'' helices, respectively . The combined use of molecular modeling and single amino acid mutagenesis is thus a useful approach for the characterization of conformational epitopes recognized by autoantibodies. J Biol Chem, 2004 Dec 3, 279(49), 50962 - 8 Epub 2004 Dec 3. The {URE3} yeast prion results from protein aggregates that differ from amyloid filaments formed in vitro; Ripaud L et al.; The {URE3} yeast prion is a self-propagating inactive form of the Ure2 protein . Ure2p is composed of two domains, residues 1-93, the prion-forming domain, and the remaining C-terminal part of the protein, which forms the functional domain involved in nitrogen catabolite repression . In vitro, Ure2p forms amyloid filaments that have been proposed to be the aggregated prion form found in vivo . Here we showed that the biochemical characteristics of these two species differ . Protease digestions of Ure2p filaments and soluble Ure2p are comparable when analyzed by Coomassie staining as by Western blot . However, this finding does not explain the pattern specifically observed in {URE3} strains . Antibodies raised against the C-terminal part of Ure2p revealed the existence of proteolysis sites efficiently cleaved when {URE3}, but not wild-type crude extracts, were submitted to limited proteolysis . The same antibodies lead to an equivalent digestion pattern when recombinant Ure2p (either soluble or amyloid) was analyzed in the same way . These results strongly suggest that aggregated Ure2p in {URE3} yeast cells is different from the amyloid filaments generated in vitro. J Biol Chem, 2004 Dec 10, 279(50), 52653 - 63 Epub 2004 Dec 10. An Escherichia coli MutY mutant without the six-helix barrel domain is a dimer in solution and assembles cooperatively into multisubunit complexes with DNA; Lee CY et al.; Escherichia coli MutY is an adenine and weak guanine DNA glycosylase involved in reducing the mutagenic effects of 7,8-dihydro-8-oxoguanine (GO) . MutY contains three structural domains: an iron-sulfur module, a six-helix barrel module with the helix-hairpin-helix motif, and a C-terminal domain . Here, we demonstrate that the mutant MutY(Delta26-134), which lacks the six-helix barrel domain, cannot complement the mutator phenotype of a mutY mutant in vivo . However, the mutant can still bind DNA and has weak catalytic activity at high enzyme concentrations . The mutant is a dimer in solution and assembled into two and multiple (up to five) complexes with 20- and 44-bp DNA fragments, respectively, in a concentration-dependent manner . Higher order complexes with DNA substrates containing A/GO mismatches were formed at lower protein concentrations than with the A/G mismatch and homoduplex DNA . Measurement of equilibrium binding using fluorescence anisotropy showed that the mutant protein retains some specificity for A/GO-containing DNA substrates and that the binding event is highly cooperative . This is consistent with the MutY structure determined, which indicates that GO specificity is contributed by both the six-helix barrel and C-terminal domains . The nonspecific binding of MutY(Delta26-134) to DNA suggests a model in which the specific binding of mismatched DNA by MutY involves sequential interactions, in which one MutY molecule scans the DNA and enhances binding of another MutY molecule to the A/GO mismatch. J Biol Chem, 2004 Dec 17, 279(51), 53451 - 7 Epub 2004 Sep 28. The active site of a lon protease from Methanococcus jannaschii distinctly differs from the canonical catalytic Dyad of Lon proteases; Im YJ et al.; ATP-dependent Lon proteases catalyze the degradation of various regulatory proteins and abnormal proteins within cells . Methanococcus jannaschii Lon (Mj-Lon) is a homologue of Escherichia coli Lon (Ec-Lon) but has two transmembrane helices within its N-terminal ATPase domain . We solved the crystal structure of the proteolytic domain of Mj-Lon using multiwavelength anomalous dispersion, refining it to 1.9-angstroms resolution . The structure displays an overall fold conserved in the proteolytic domain of Ec-Lon; however, the active site shows uniquely configured catalytic Ser-Lys-Asp residues that are not seen in Ec-Lon, which contains a catalytic dyad . In Mj-Lon, the C-terminal half of the beta4-alpha2 segment is an alpha-helix, whereas it is a beta-strand in Ec-Lon . Consequently, the configurations of the active sites differ due to the formation of a salt bridge between Asp-547 and Lys-593 in Mj-Lon . Moreover, unlike Ec-Lon, Mj-Lon has a buried cavity in the region of the active site containing three water molecules, one of which is hydrogen-bonded to catalytic Ser-550 . The geometry and environment of the active site residues in Mj-Lon suggest that the charged Lys-593 assists in lowering the pK(a) of the Ser-550 hydroxyl group via its electrostatic potential, and the water in the cavity acts as a proton acceptor during catalysis . Extensive sequence alignment and comparison of the structures of the proteolytic domains clearly indicate that Lon proteases can be classified into two groups depending on active site configuration and the presence of DGPSA or (D/E)GDSA consensus sequences, as represented by Ec-Lon and Mj-Lon. Acta Pharmacol Sin, 2004 Oct, 25(10), 1292 - 8 Production of neutralizing monoclonal antibody against human vascular endothelial growth factor receptor II; Li R et al.; AIM: To prepare neutralizing monoclonal antibody (mAb) against extracellular immunoglobulin (Ig)-like domain III of vascular endothelial growth factor receptor KDR and study its biological activity . METHODS: Soluble KDR Ig domain III (KDR-III) fusion protein was expressed in E Coli and purified from the bacterial periplasmic extracts via an affinity chromatography . Monoclonal antibodies against KDR-III were prepared by hybridoma technique . ELISA and FACS analysis were used to identify its specificity . Immunoprecipitation and {3H}-thymidine incorporation assay were also used to detect the activity of anti-KDR mAb blocking the phosphorylation of KDR tyrosine kinase receptor and the influence on vascular endothelial growth factor-induced mitogenesis of human endothelial cells . RESULTS: A monoclonal antibody, Ycom1D3 (IgG1), was generated from a mouse immunized with the recombinant KDR-III protein . Ycom1D3 bound specifically to both the soluble KDR-III and the cell-surface expressed KDR . Ycom1D3 effectively blocked VEGF/KDR interaction and inhibited VEGF-stimulated KDR activation in human endothelial cells . Furthermore, the antibody efficiently neutralized VEGF-induced mitogenesis of human endothelial cells . CONCLUSION: Our results suggest that the anti-KDR mAb, Ycom1D3, has potential applications in the treatment of cancer and other diseases where pathological angiogenesis is involved . Anal Chem, 2004 Oct 1, 76(19), 5620 - 9 General strategy for biosensor design and construction employing multifunctional surface-tethered components; Medintz IL et al.; Biosensors function by reversibly linking bioreceptor-target analyte binding with closely integrated signal generation and can either continuously monitor analyte concentrations or be returned to baseline readout values by removal of analyte . We present an approach for producing fully reversible, reagentless, self-assembling biosensors on surfaces . In the prototype biosensor, quencher-dye-labeled biotin-linked E . coli maltose binding protein (MBP) bound in a specific orientation to a NeutrAvidin-coated surface is employed as a bioreceptor . To complete sensor formation, a modular tether arm consisting of a flexible biotinylated DNA oligonucleotide, a fluorescence resonance energy-transfer (FRET) donor dye, and a distal beta-cyclodextrin (beta-CD) analyte analogue is bound in an equimolar amount to the same surface by means of DNA-directed immobilization . After self-assembly, a baseline level of FRET quenching is observed due to specific interaction between the beta-CD of the flexible tether arm and the sugar binding site of MBP, which brings the two dyes into proximity . Addition of the target analyte, the nutrient maltose, displaces the linked beta-CD-dye of the DNA-based tether arm, and a concentration-dependent change in FRET results . Biosensor sensitivity and dynamic range can be controlled by either using MBP variants having different binding constants or by binding of modulator DNA oligonucleotides that are complementary to the flexible DNA tether . The sensor can be regenerated and returned to baseline quenching levels by washing away analyte . A complex set of interactions apparently exists on the sensing surface that may contribute to sensor behavior and range . This approach may represent a general way to assemble a wide range of useful biosensors. Mol Biol (Mosk), 2004 Jul-Aug, 38(4), 713 - 6 {Construction of pMH, a convenient Escherichia coli protein expression vector}; Sensing of remote oxyanion binding at the DNA binding domain of the molybdate-dependent transcriptional regulator et al.; Division of Biological Chemistry and Molecular Microbiology, School of Life Sciences, University of Dundee, Dundee, UKThe molybdate-dependent transcriptional regulator ModE of Escherichia coli displays a large (50%) quenching of its intrinsic tryptophan fluorescence on binding molybdate . The changes in fluorescence have been exploited to analyse the binding of molybdate to ModE . Utilising site-directed mutagenesis, a series of phenylalanine substitutions for the three tryptophans of ModE (Trp49, Trp131 and Trp186) have been constructed, to yield three mono-Trp-containing derivatives . This has allowed an assessment to be made of the contribution of each of the three tryptophans to the spectral changes observed on binding molybdate; these are most distinctive for Trp186 . Linkage between the DNA-binding and molybdate-binding sites (some 55 angstroms apart) is shown by (a) the small, but definite, effect of molybdate on the fluorescence of Trp49 which is located at the DNA-binding winged helix-turn-helix domain, and (b) the finding that the binding of either ligand is enhanced in the presence of the other . The studies demonstrate that the mono-Trp derivatives of ModE could be useful tools with which to study the signal transduction processes specifically associated with molybdate-dependent transcriptional regulation and that this approach may have wider implications for analysis of other regulated systems . Genetics, 2004 Sep, 168(1), 541 - 6 Stress and survival of aging Escherichia coli rpoS colonies; Saint-Ruf C et al.; In Escherichia coli, the expression of the RpoS regulon is known to be crucial for survival in liquid cultures during stationary phase . By measuring cell viability and by transcriptome analysis, here we show that rpoS cells as well as wild-type cells survive when they form colonies on solid media. Biophys J, 2004 Oct, 87(4), 2683 - 90 LexA-DNA bond strength by single molecule force spectroscopy; Kuhner F et al.; The SOS system of Escherichia coli is coordinated by two proteins: LexA, a repressor protein of several unlinked genes, and the coprotease RecA . As known to date LexA controls 31 genes with slightly different DNA binding motifs allowing for a variable degree of repression from one gene to the other . Besides the SOS system LexA plays an important role in the regulation of transcription . The protein regulates transcription by using particular motifs to bind DNA, the helix-turn-helix motif . Here, we employed AFM-based single molecule force spectroscopy to characterize the interaction of LexA protein with two different DNA motifs: recA and yebG . We measured the dissociation rates to be 0.045 s(-1) for recA and 0.13 s(-1) for yebG, respectively, which is in accordance with the predicted higher affinity between LexA-recA compared to LexA-yebG . The widths of the binding potentials were determined to be 5.4 +/- 1 angstroms and 4.9 +/- 0.5 angstroms, respectively . This short-ranged potential is characteristic for a stiff hydrogen-bonding network between protein and DNA . The unbinding occurs in a breakup rather than a gradual sliding . Biophys J, 2004 Dec, 87(6), 3922 - 33 Epub 2004 Dec. Effects of two familial hypertrophic cardiomyopathy mutations in alpha-tropomyosin, Asp175Asn and Glu180Gly, on the thermal unfolding of actin-bound tropomyosin; Kremneva E et al.; Differential scanning calorimetry was used to investigate the thermal unfolding of native alpha-tropomyosin (Tm), wild-type alpha-Tm expressed in Escherichia coli and the wild-type alpha-Tm carrying either of two missense mutations associated with familial hypertrophic cardiomyopathy, D175N or E180G . Recombinant alpha-Tm was expressed with an N-terminal Ala-Ser extension to substitute for the essential N-terminal acetylation of the native Tm . Native and Ala-Ser-Tm were indistinguishable in our assays . In the absence of F-actin, the thermal unfolding of Tm was reversible and the heat sorption curve of Tm with Cys-190 reduced was decomposed into two separate calorimetric domains with maxima at approximately 42 and 51 degrees C . In the presence of phalloidin-stabilized F-actin, a new cooperative transition appears at 46-47 degrees C and completely disappears after the irreversible denaturation of F-actin . A good correlation was found to exist between the maximum of this peak and the temperature of half-maximal dissociation of the F-actin/Tm complex as determined by light scattering experiments . We conclude that Tm thermal denaturation only occurs upon its dissociation from F-actin . In the presence of F-actin, D175N alpha-Tm shows a melting profile and temperature dependence of dissociation from F-actin similar to those for wild-type alpha-Tm . The actin-induced stabilization of E180G alpha-Tm is significantly less than for wild-type alpha-Tm and D175N alpha-Tm, and this property could contribute to the more severe myopathy phenotype reported for this mutation. Bioorg Med Chem Lett, 2004 Nov 1, 14(21), 5309 - 12 Substrate analogs for the investigation of deoxyxylulose 5-phosphate reductoisomerase inhibition: synthesis and evaluation; Phaosiri C et al.; Deoxyxylulose 5-phosphate (DXP) analogs were synthesized and evaluated as alternative substrates and inhibitors of recombinant Synechocystis PCC6803 DXP reductoisomerase (DXR; EC 1.1.1.267) . Five of the compounds tested (1,2-dideoxy-D-threo-3-hexulose 6-phosphate, 1-deoxy-l-ribulose 5-phosphate, 2S,3R-dihydroxybutyramide 4-phosphate, 4S-hydroxypentan-2-one 5-phosphate, and 3S-hydroxypentan-2-one 5-phosphate) acted as relatively weak competitive inhibitors when compared to fosmidomycin . A sixth compound, 3R,4S-dihydroxy-5-oxohexylphosphonic acid, served as an alternate substrate, as has recently been reported for the same compound with Escherichia coli DXR. J Am Chem Soc, 2004 Oct 6, 126(39), 12477 - 91 Spectroscopic and computational studies of the azide-adduct of manganese superoxide dismutase: definitive assignment of the ligand responsible for the low-temperature thermochromism; Jackson TA et al.; A variety of spectroscopic and computational techniques have been used to examine the thermochromic transition previously reported for the oxidized state of Mn-dependent superoxide dismutase from E . coli in the presence of substrate analog azide (N(3)-Mn(3+)SOD).{Whittaker, M . M.; Whittaker, J . W . Biochemistry 1996, 35, 6762-6770.} Although previous spectroscopic studies had shown that this thermochromic event corresponds to a change in coordination number of the active-site Mn(3+) ion from 6 to 5 as temperature is increased, the ligand that dissociates in this conversion had yet to be identified . Through the use of electronic absorption, circular dichroism (CD), and magnetic CD (MCD) spectroscopies, both d-->d and ligand-to-metal charge-transfer (LMCT) transition energies have been determined for native Mn(3+)SOD (possessing a five-coordinate Mn(3+) center) and Y34F N(3)-Mn(3+)SOD (forming a six-coordinate N(3)-Mn(3+) adduct at all temperatures) . These two systems provide well-defined reference points from which to analyze the absorption and CD data obtained for N(3)-Mn(3+)SOD at room temperature (RT) . Comparison of excited-state spectroscopic data reveals that Mn(3+)SOD and RT N(3)-Mn(3+)SOD exhibit virtually identical d-->d transition energies, suggesting that these two species possess similar geometric and electronic structures and, thus, that azide does not actually coordinate to the active-site Mn(3+) ion at RT . However, resonance Raman spectra of both N(3)-Mn(3+)SOD and Y34F N(3)-Mn(3+)SOD at 0 degrees C exhibit azide-related vibrations, indicating that azide does interact with the active site of the native enzyme at this temperature . To gain further insight into the nature of the azide/Mn(3+) interaction in RT N(3)-Mn(3+)SOD, several viable active-site models designed to promote either dissociation of coordinated solvent, Asp167, or azide were generated using DFT computations . By utilizing the time-dependent DFT method to predict absorption spectra for these models of RT N(3)-Mn(3+)SOD, we demonstrate that only azide dissociation is consistent with experimental data . Collectively, our spectroscopic and computational data provide evidence that the active site of N(3)-Mn(3+)SOD at RT exists in a dynamic equilibrium, with the azide molecule either hydrogen-bonded to the second-sphere Tyr34 residue or coordinated to the Mn(3+) ion . These results further highlight the role that second-sphere residues, especially Tyr34, play in tuning substrate (analog)/metal ion interactions. J Am Chem Soc, 2004 Oct 6, 126(39), 12316 - 24 Magnetic circular dichroism and cobalt(II) binding equilibrium studies of Escherichia coli methionyl aminopeptidase; Larrabee JA et al.; Equilibrium dialysis of methionyl aminopeptidase from Escherichia coli (EcMetAP) monitored by atomic absorption spectrometry and magnetic circular dichroism (MCD) shows that the enzyme binds up to 1.1 +/- 0.1 equiv of Co(2+) in the metal concentration range likely to be found in vivo . The dissociation constant, K(d), is estimated to be between 2.5 and 4.0 microM . Analysis of the temperature and magnetization behavior of the two major peaks in the MCD spectrum at 495 and 567 nm suggests that these transitions arise from Co(2+) with different ground states . Ligand field calculations using AOMX are used to assign the 495 nm peak to Co(2+) in the 6-coordinate binding site and the 567 nm peak to Co(2+) in the 5-coordinate site . This is further supported by the fact that the binding affinity of the Co(2+) associated with the 567 nm peak is enhanced when the pH is increased from 7.5 to 9.0, consistent with having an imidazole ligand from a histidine amino acid residue . On the basis of the MCD intensities, it is estimated that, when the 5-coordinate site is fully occupied, 0.1 equiv of cobalt is in the 6-coordinate site . Even when the cobalt concentration is very low, there is a small fraction of binuclear sites in EcMetAP formed through cooperative binding between the 5- and 6-coordinate Co(2+) ions . The magnetization behavior of the 6-coordinate Co(2+) MCD peak is consistent with an isolated pseudo-Kramer doublet ground state, suggesting that the cobalt ions in the binuclear sites are not magnetically coupled. Adv Biochem Eng Biotechnol, 2004, 91, 1 - 49 Metabolic flux analysis based on 13C-labeling experiments and integration of the information with gene and protein expression patterns; Shimizu K; The recent progress on metabolic systems engineering was reviewed, in particular focusing on the metabolic flux analysis (MFA) based on the isotopomer distribution obtained using NMR and/or GC-MS . After the brief explanation of how to estimate the metabolic flux distribution (MFD) based on 13C-labeling experiments, the metabolic regulation analysis was made based on the protein expression patterns obtained by two-dimensional electrophoresis (2DE) together with enzyme activity data, and the gene expression patterns obtained by RT-PCR analysis . The particular application was considered for Escherichia coli . The effect of culture conditions such as different carbon sources (glucose, gluconate, glycerol, acetate, etc.) and different dissolved oxygen (DO) concentration, etc . on the metabolism was investigated . The effect of some single-gene knockout such as pgi-, pyk-, and gnd- was also investigated . It was found to be quite useful to integrate the information obtained from metabolic flux analysis, gene and protein expressions as well as intracellular metabolite concentrations to understand the overall picture of metabolic regulation. Adv Biochem Eng Biotechnol, 2004, 90, 135 - 49 Cell-free protein synthesis systems: increasing their performance and applications; Nakano H et al.; The Escherichia coli cell-free protein synthesis system can now be used for various proteins that need special requirements, such as disulfide bond formation between intra- and inter-molecules, hetero-dimerization, and specific chaperons . In addition, a novel protein library construction method termed "SIMPLEX" has been developed . Some applications with SIMPLEX are described . A highly efficient eukaryotic cell-free translation system using wheat germ extract has also been developed . An advanced framework for a rapid eukaryotic gene expression and analysis is shown. J Biomol NMR, 2004 Sep, 30(1), 37 - 45 A novel way of amino acid-specific assignment in (1)H-(15)N HSQC spectra with a wheat germ cell-free protein synthesis system; Morita EH et al.; For high-throughput protein structural analyses, it is indispensable to develop a reliable protein overexpression system . Although many protein overexpression systems, such as ones utilizing E . coli cells, have been developed, a lot of proteins functioning in solution still were synthesized as insoluble forms . Recently, a novel wheat germ cell-free protein synthesis system was developed, and many of such proteins were synthesized as soluble forms . This means that the applicability of this protein synthesis method to determination of the functional structures of soluble proteins . In our previous work, we synthesized (15)N-labeled proteins with this wheat germ cell-free system, and confirmed this applicability on the basis of the strong similarity between the (1)H-(15)N HSQC spectra for native proteins and the corresponding ones for synthesized ones.In this study, we developed a convenient and reliable method for amino acid selective assignment in (1)H-(15)N HSQC spectra of proteins, using several inhibitors for transaminases and glutamine synthase in the process of protein synthesis . Amino acid selective assignment in (1)H-(15)N HSQC spectra is a powerful means to monitor the features of proteins, such as folding, intermolecular interactions and so on . This is also the first direct experimental evidence of the presence of active transaminases and glutamine synthase in wheat germ extracts. Proc Natl Acad Sci U S A, 2004 Oct 5, 101(40), 14396 - 401 Epub 2004 Sep 27. Long-range intramolecular signaling in a tRNA synthetase complex revealed by pre-steady-state kinetics; Uter NT et al.; Pre-steady-state kinetic studies of Escherichia coli glutaminyl-tRNA synthetase conclusively demonstrate the existence of long-distance pathways of communication through the protein-RNA complex . Measurements of aminoacyl-tRNA synthesis reveal a rapid burst of product formation followed by a slower linear increase corresponding to k(cat) . Thus, a step after chemistry but before regeneration of active enzyme is rate-limiting for synthesis of Gln-tRNA(Gln) . Single-turnover kinetics validates these observations, confirming that the rate of the chemical step for tRNA aminoacylation (k(chem)) exceeds the steady-state rate by nearly 10-fold . The concentration dependence of the single-turnover reaction further reveals that the glutamine K(d) is significantly higher than the steady-state K(m) value . The separation of binding from catalytic events by transient kinetics now allows precise interpretation of how alterations in tRNA structure affect the aminoacylation reaction . Mutation of U35 in the tRNA anticodon loop decreases k(chem) by 30-fold and weakens glutamine binding affinity by 20-fold, demonstrating that the active-site configuration depends on enzyme-tRNA contacts some 40 A distant . By contrast, mutation of the adjacent G36 has very small effects on k(chem) and K(d) for glutamine . Together with x-ray crystallographic data, these findings allow a comparative evaluation of alternative long-range signaling pathways and lay the groundwork for systematic exploration of how induced-fit conformational transitions may control substrate selection in this model enzyme-RNA complex. J Biol Chem, 2004 Dec 3, 279(49), 50915 - 22 Epub 2004 Dec 3. Hiding behind hydrophobicity . Transmembrane segments in mass spectrometry; Eichacker LA et al.; Proteomics of membrane proteins is essential for the understanding of cellular function . However, mass spectrometric analysis of membrane proteomes has been less successful than the proteomic determination of soluble proteins . To elucidate the mystery of transmembrane proteins in mass spectrometry, we present a detailed statistical analysis of experimental data derived from chloroplast membranes . This approach was further accomplished by the analysis of the Arabidopsis thaliana proteome after in silico digestion . We demonstrate that both the length and the hydrophobicity of the proteolytic fragments containing transmembrane segments are major determinants for detection by mass spectrometry . Based on a comparative analysis, we discuss possibilities to overcome the problem and provide possible protocols to shift the hydrophobicity of transmembrane segment-containing peptides to facilitate their detection. J Mol Biol, 2004 Oct 15, 343(2), 493 - 510 Structural determinants of proton blockage in aquaporins; Chakrabarti N et al.; Aquaporins are an important class of membrane channels selective for water and linear polyols but impermeable to ions, including protons . Recent computational studies have revealed that the relay of protons through the water-conduction pathway of aquaporin channels is opposed by a substantial free energy barrier peaking at the signature NPA motifs . Here, free-energy simulations and continuum electrostatic calculations are combined to examine the nature and the magnitude of the contribution of specific structural elements to proton blockage in the bacterial glycerol uptake facilitator, GlpF . Potential of mean-force profiles for both hop and turn steps of structural diffusion in the narrow pore are obtained for artificial variants of the GlpF channel in which coulombic interactions between the pore contents and conserved residues Asn68 and Asn203 at the NPA signature motifs, Arg206 at the selectivity filter, and the peptidic backbone of the two half-helices M3 and M7, which are arranged in head-to-head fashion around the NPA motifs, are turned off selectively . A comparison of these results with electrostatic energy profiles for the translocation of a probe cation throughout the water permeation pathway indicates that the free-energy profile for proton movement inside the narrow pore is dominated by static effects arising from the distribution of charged and polar groups of the channel, whereas dielectric effects contribute primarily to opposing the access of H+ to the pore mouths (desolvation penalty) . The single most effective way to abolish the free-energy gradients opposing the movement of H+ around the NPA motif is to turn off the dipole moments of helices M3 and M7 . Mutation of either of the two NPA Asn residues to Asp compensates for charge-dipole and dipole-dipole effects opposing the hop and turn steps of structural diffusion, respectively, and dramatically reduces the free energy barrier of proton translocation, suggesting that these single mutants could leak protons. J Mol Biol, 2004 Oct 15, 343(2), 305 - 12 Crystallographic structure of the nuclease domain of 3'hExo, a DEDDh family member, bound to rAMP; Cheng Y et al.; A human 3'-5'-exoribonuclease (3'hExo) has recently been identified and shown to be responsible for histone mRNA degradation . Functionally, 3'hExo and a stem-loop binding protein (SLBP) target opposite faces of a unique highly conserved stem-loop RNA scaffold towards the 3' end of histone mRNA, which is composed of a 6 bp stem and a 4 nt loop, followed by an ACCCA sequence . Its Caenorhabditis elegans homologue, ERI-1, has been shown to degrade small interfering RNA in vitro and to function as a negative regulator of RNA interference in neuronal cells . We have determined the structure of the nuclease domain (Nuc) of 3'hExo complexed with rAMP in the presence of Mg2+ at 1.6 A resolution . The Nuc domain adopts an alpha/beta globular fold, with four acidic residues coordinating a binuclear metal cluster within the active site, whose topology is related to DEDDh exonuclease family members, despite a very low level of primary sequence identity . The two magnesium cations in the Nuc active site are coordinated to D134, E136, D234 and D298, and together with H293, which can potentially act as a general base, provide a platform for hydrolytic cleavage of bound RNA in the 3' --> 5' direction . The bound rAMP is positioned within a deep active-site pocket, with its purine ring close-packed with the hydrophobic F185 and L189 side-chains and its sugar 2'-OH and 3'-OH groups hydrogen bonded to backbone atoms of Nuc . There are striking similarities between the active sites of Nuc and epsilon186, an Escherichia coli DNA polymerase III proofreading domain, providing a common hydrolytic cleavage mechanism for RNA degradation and DNA editing, respectively. Toxicol Lett, 2004 Nov 2, 153(2), 267 - 72 Enhanced expression of human cytochrome P450 1A2 by co-expression with human molecular chaperone Hsp70; Ahn T et al.; Cytochrome P450 (CYP) 1A2 is of great interest because of its important roles in the oxidation of numerous drugs and carcinogens . Hsp70, a molecular chaperone in human, is known to assist the correct folding of unfolded proteins . To achieve high yield of recombinant human CYP1A2 in Escherichia coli, the CYP1A2 encoding gene was co-expressed with the chaperone Hsp70 under the control of an inducible tac promoter in bicistronic format . Expression level of CYP1A2 in the bicistronic construct reached up to 410 nmol (lculture)(-1) within 16 h at 37 degrees C, which is approximately 2.7-fold increase compared to the expression yield of CYP1A2 alone without Hsp70 . The present over-expression system may be useful for rapid production of large amounts of active CYP1A2 in E . coli. FEMS Microbiol Lett, 2004 Oct 1, 239(1), 131 - 8 Identification and molecular characterization of the gene encoding coli surface antigen 20 of enterotoxigenic Escherichia coli; Valvatne H et al.; Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhea among children living in developing countries and of travelers' diarrhea . Current ETEC vaccine designs aim to induce an anti-colonizing immunity by including the ETEC surface colonization factor antigens . We isolated and characterized the structural gene of the coli surface antigen 20 (CS20) . CS20 has an N-terminal amino acid sequence similar to that of CS18 . We therefore used a DNA fragment carrying the CS18 fotA gene as a probe in a hybridization assay to detect the corresponding gene in a CS20-positive strain isolated from an Indian child . Cross hybridizing DNA was isolated and found to contain an open reading frame encoding a polypeptide of 195 amino acids, including a 22 amino acid signal peptide . The gene, which we named csnA, shows a high degree of identity to the major fimbrial subunits of CS12, CS18 and F6 (also referred to as 987P), a CS of porcine ETEC . The coding region of csnA was inserted into an expression system to generate a polypeptide confirmed to be CS20 by Western blot . A CS20 colony hybridization assay using a DNA probe derived from csnA was developed. FEMS Microbiol Lett, 2004 Oct 1, 239(1), 33 - 9 Expression and purification of recombinant methylated HBHA in Mycobacterium smegmatis; Delogu G et al.; The Heparin-Binding Haemagglutinin (HBHA) is a mycobacterial adhesin involved in the dissemination of Mycobacterium tuberculosis from the site of primary infection and a potential candidate for the development of a new vaccine against tuberculosis . Methylation of HBHA is a novel post-translational event that imparts important immunological properties to the protein . Since recombinant HBHA expressed in Escherichia coli is not methylated, we investigated the possibility of producing recombinant methylated HBHA in fast growing mycobacteria for use in immunological and biochemical studies . The complete coding sequence of HBHA was cloned in the plasmid pMV206, under the control of a strong promoter (hsp60) or its own promoter . The constructs generated were electroporated into Mycobacterium smegmatis and the recombinant strains obtained were analyzed for the presence of the HBHA protein using the anti-HBHA monoclonal antibodies D2 and E4 . Our results indicate that expression of high amounts of intact protein can be toxic for the mycobacteria, that methylated HBHA can be obtained in M . smegmatis only when using a promoter sequence weaker than hsp60 and that the expression of the complete structural gene is required in order to obtain methylated HBHA . We constructed a recombinant M . smegmatis strain (pMV3-38) that expresses a histidine-tagged methylated HBHA that can be easily purified . The use of fast-growing strains of M . smegmatis to obtain significant amounts of purified HBHA protein within a short timeframe, should be an effective strategy for the evaluation of a new HBHA-based vaccine candidate for tuberculosis. Biochim Biophys Acta, 2004 Oct 1, 1702(1), 121 - 4 Crystallization and preliminary X-ray crystallographic analysis of strictosidine synthase from Rauvolfia: the first member of a novel enzyme family; Ma X et al.; Strictosidine synthase is a central enzyme involved in the biosynthesis of almost all plant monoterpenoid indole alkaloids . Strictosidine synthase from Rauvolfia serpentina was heterologously expressed in Escherichia coli . Crystals of the purified recombinant enzyme have been obtained by the hanging-drop technique at 303 K with potassium sodium tartrate tetrahydrate as precipitant . The crystals belong to the space group R3 with cell dimensions of a=b=150.3 A and c=122.4 A . Under cryoconditions (120 K), the crystals diffract to about 2.95 A. Am J Surg, 2004 Sep, 188(3), 288 - 93 Role of a hyaluronic-acid derivative in preventing surgical adhesions and abscesses related to dropped bile and gallstones in an experimental model; Aytekin FO et al.; BACKGROUND: Despite its advantages, iatrogenic gallbladder perforation with resultant spillage of bile and gallstones is not uncommon during laparoscopic cholecystectomy . Although this is not generally considered a significant problem, spilled gallstones may cause problems even years after the operation . Hyaluronic acid has been introduced into clinical practice and successfully used to decrease postoperative adhesions after abdominopelvic surgery . In this study, we investigated the effectiveness of a hyaluronic-acid derivative in preventing complications related to spilled gallstones and bile leakage in an experimental study . METHODS: In 60 Wistar-Albino rats, an upper-midline abdominal incision was made, and the rats were divided into 5 groups (n = 12 in each group) as follows: group I = laparotomy alone; group II = laparotomy and intraperitoneal instillation of sterile bile plus gallstones; group III = laparotomy and instillation of infected bile and gallstones; group IV = laparotomy and instillation of sterile bile and gallstones plus hyaluronic acid; and group V = laparotomy and instillation of infected bile and gallstones plus HA . A second-look laparotomy was performed on postoperative day 10 to assess intraperitoneal adhesions and abscesses . Intraperitoneal adhesions were scored, and breaking strengths of gallstones were measured . RESULTS: Adhesion scores were significantly higher in groups II and III compared with groups I, IV, and V (P < 0.05) . There was a statistically significant decrease in breaking strengths and adhesion scores in groups IV and V compared with groups II and III (P < 0.001) . CONCLUSIONS: Whether infected or not, spilled gallstones and bile caused postoperative adhesions in this experimental model . An HA derivative significantly prevented postoperative adhesions and decreased breaking strengths . Further clinical studies are needed to validate these findings . Ultramicroscopy, 2004 Nov, 101(2-4), 231 - 40 Tip to substrate distances in STM imaging of biomolecules; Alliata D et al.; STM images of single biomolecules adsorbed on conductive substrates do not reproduce the expected physical height, which generally appears underestimated . This may cause the tip to interfere with the soft biological sample during the imaging scans . Therefore, a key requirement to avoid invasive STM imaging is the knowledge, and the control, of the initial tip to substrate distance . This is connected to the setting of the tunnelling current and applied voltage, which define a tunnelling resistance . The height of the STM tip was measured by calibrating the tunnelling resistance, as a function of its vertical displacement until establishing a mechanical contact . At a tunnelling resistance of 4 x 10(9)Omega, distances of about 3 and 6 nm are estimated when flat Au substrates are imaged in water and in air, respectively . On such a ground, the relevance of the starting tip-substrate distance in determining a non-invasive imaging has been investigated for a plastocyanin mutant chemisorbed on Au(111) electrodes . At tunnelling distances sufficient to overcome the physical height of the imaged biomolecules, their lateral dimensions are found to be consistent with the crystallography, whereas they are significantly broadened for smaller distances. Mutat Res, 2004 Oct 4, 554(1-2), 193 - 203 Positive selection for loss of RpoS function in Escherichia coli; Chen G et al.; Though RpoS, an alternative sigma factor, is required for survival and adaptation of Escherichia coli under stress conditions, many strains have acquired independent mutations in the rpoS gene . The reasons for this apparent selective loss and the nature of the selective agent are not well understood . In this study, we found that some wild type strains grow poorly in succinate minimal media compared with isogenic strains carrying defined RpoS null mutations . Using an rpoS+ strain harboring an operon lacZ fusion to the highly-RpoS dependent osmY promoter as an indicator strain, we tested if this differential growth characteristic could be used to selectively isolate mutants that have lost RpoS function . All isolated (Suc+) mutants exhibited attenuated beta-galactosidase expression on indicator media suggesting a loss in either RpoS or osmY promoter function . Because all Suc+ mutants were also defective in catalase activity, an OsmY-independent, RpoS-regulated function, it was likely that RpoS activity was affected . To confirm this, we sequenced PCR-amplified products containing the rpoS gene from 20 independent mutants using chromosomal DNA as a template . Sequencing and alignment analyses confirmed that all isolated mutants possessed mutated alleles of the rpoS gene . Types of mutations detected included single or multiple base deletions, insertions, and transversions . No transition mutations were identified . All identified point mutations could, under selection for restoration of beta-galactosidase, revert to rpoS+ . Revertible mutation of the rpoS gene can thus function as a genetic switch that controls expression of the regulon at the population level . These results may also help to explain why independent laboratory strains have acquired mutations in this important regulatory gene. Mutat Res, 2004 Oct 4, 554(1-2), 149 - 57 RecN and RecG are required for Escherichia coli survival of Bleomycin-induced damage; Kosa JL et al.; The sensitivity of a panel of DNA repair-defective bacterial strains to BLM was investigated . Escherichia coli recA cells were far more sensitive than were uvrA, dam-3, and mutM mutY strains, underscoring the importance of RecA to survival . Strains recBCD and recN, which lack proteins required for double strand break (DSB) repair, were highly sensitive to BLM, while recF cells were not . The requirement for DSB-specific enzymes supports the hypothesis that DSBs are the primary cause of bleomycin cytotoxicity . The acute sensitivity of recN cells was comparable to that of recA, implying a central role for the RecN protein in BLM lesion repair . The Holliday junction processing enzymes RecG and RuvC were both required for BLM survival . The recG ruvC double mutant was no more sensitive than either mutation alone, suggesting that both enzymes participate in the same pathway . Surprisingly, ruvAB cells were no more sensitive than wildtype, implying that RuvC is able to perform its role without RuvAB . This observation contrasts with current models of recombination in which RuvA, B, and C function as a single complex . The most straightforward explanation of these results is that DSB repair involves a structure that serves as a good substrate for RecG, and not RuvAB. J Struct Biol, 2004 Sep, 147(3), 315 - 26 Normal mode based flexible fitting of high-resolution structure into low-resolution experimental data from cryo-EM; Tama F et al.; A new method for the flexible fitting of high-resolution structures into low-resolution maps of macromolecular complexes from electron microscopy has been recently described in applications to simulated electron density maps . This method uses a linear combination of low-frequency normal modes in an iterative manner to deform the structure optimally to conform to the low-resolution electron density map . Gradient-following techniques in the coordinate space of collective normal modes are used to optimize the overall correlation coefficient between computed and measured electron densities . With this approach, multi-scale flexible fitting can be performed using all-atoms or Calpha atoms . In this paper, illustrative studies of normal mode based flexible fitting to experimental cryo-EM maps are presented for three different systems . Large, functionally relevant conformational changes for elongation factor G bound to the ribosome, Escherichia coli RNA polymerase and cowpea chlorotic mottle virus are elucidated as the result of the application of NMFF from high-resolution structures to cryo-electron microscopy maps . J Struct Biol, 2004 Sep, 147(3), 283 - 90 Dynamics of EF-G interaction with the ribosome explored by classification of a heterogeneous cryo-EM dataset; Gao H et al.; A method of supervised classification using two available structure templates was applied to investigate the possible heterogeneity existing in a large cryo-EM dataset of an Escherichia coli 70S ribosome-EF-G complex . Two subpopulations showing the ribosome in distinct conformational states, related by a ratchet-like rotation of the 30S subunit with respect to the 50S subunit, were extracted from the original dataset . The possible presence of additional intermediate states is discussed . Biochim Biophys Acta, 2004 Aug 30, 1684(1-3), 38 - 45 Highly polymorphic repeat region in the CETP promoter induces unusual DNA structure; Lira ME et al.; Genetic variation in the human cholesteryl ester transfer protein (CETP) promoter is associated with HDL cholesterol levels and cardiovascular disease with much of the genetic variation in CETP attributed to the promoter region . In this region, there are several single nucleotide polymorphisms as well as a variable length tandem repeat located 1946 base pairs upstream of the CETP transcription start that is highly polymorphic with respect to both length and sequence . There are more than 10 different long alleles and these vary in their repeat structure . We find that the short allele of this repeat is associated with high HDL cholesterol levels in vivo (P<0.0001) . In males, this association is independent of the nearby -629 polymorphism . In addition, the variable length GAAA repeat can stimulate an adjacent GGGGA repeat to form a structure that hinders DNA amplification and sequencing . This structure also has an effect in vivo as shown by orientation effects and cloning efficiency in Escherichia coli. Biochim Biophys Acta, 2004 Sep 1, 1701(1-2), 129 - 32 Vinorine synthase from Rauvolfia: the first example of crystallization and preliminary X-ray diffraction analysis of an enzyme of the BAHD superfamily; Ma X et al.; Crystals of vinorine synthase (VS) from medicinal plant Rauvolfia serpentina expressed in Escherichia coli have been obtained by the hanging-drop technique at 305 K with ammonium sulfate and PEG 400 as precipitants . The enzyme is involved in the biosynthesis of the antiarrhythmic drug ajmaline and is a member of the BAHD superfamily of acyltransferases . So far, no three-dimensional structure of a member of this enzyme family is known . The crystals belong to the space group P2(1)2(1)2(1) with cell dimensions of a=82.3 A, b=89.6 A and c=136.2 A . Under cryoconditions (120 K), a complete data set up to 2.8 A was collected at a synchrotron source. Biochim Biophys Acta, 2004 Sep 1, 1701(1-2), 49 - 59 Probing a hydrogen bond pair and the FAD redox properties in the proline dehydrogenase domain of Escherichia coli PutA; Baban BA et al.; The PutA flavoprotein from Escherichia coli combines DNA-binding, proline dehydrogenase (PRODH), and Delta(1)-pyrroline-5-carboxylate dehydrogenase (P5CDH) activities onto a single polypeptide . Recently, an X-ray crystal structure of PutA residues 87-612 was solved which identified a D370-Y540 hydrogen bond pair in the PRODH active site that appears to have an important role in shaping proline binding and the FAD redox environment . To examine the role of D370-Y540 in the PRODH active site, mutants D370A, Y540F, and D370A/Y540F were characterized in a form of PutA containing only residues 86-601 (PutA86-601) designed to mimic the known structural region of PutA (87-612) . Disruption of the D370-Y540 pair only slightly diminished k(cat), while more noticeable affects were observed in K(m) . The mutant D370A/Y540F showed the most significant changes in the pH dependence of k(cat)/K(m) and K(m) relative to wild-type PutA86-601 with an apparent pK(a) value of about 8.2 for the pH-dependent decrease in K(m) . From the pH profile of D370A/Y540F inhibition by l-tetrahydro-2-furoic acid (l-THFA), the pH dependency of K(m) in D370A/Y540F is interpreted as resulting from the deprotonation of the proline amine in the E-S complex . Replacement of D370 and Y540 produces divergent effects on the E(m) for bound FAD . At pH 7.0, E(m) values of -0.026, -0.089 and -0.042 V were determined for the two-electron reduction of bound FAD in D370A, Y540F and D370A/Y540F, respectively . The 40-mV positive shift in E(m) determined for D370A relative to wild-type PutA86-601 (E(m)=-0.066 V, pH 7.0) indicates D370 has a key role in modulating the FAD redox environment. Biochim Biophys Acta, 2004 Sep 1, 1701(1-2), 15 - 23 DNA binding properties of protein TrwA, a possible structural variant of the Arc repressor superfamily; Moncalian G et al.; Conjugative DNA processing of plasmid R388 requires the concerted action of two proteins, the relaxase-helicase TrwC and the relaxase enhancer TrwA . TrwA can be aligned with DNA binding proteins belonging to the ribbon-helix-helix (RHH) protein family . To further analyse TrwA function, the structural domains of the protein have been identified and dissected by limited proteolysis . Two stable domains were found that resulted to be, according to DNA binding experiments and oligomerization analysis, an N-terminal DNA binding domain and a C-terminal tetramerization domain . Using the three-dimensional structure of the Arc repressor as a guide, it was possible to model TrwA DNA binding site with atomic detail . As a result, TrwA polar amino acids Q8, R10 and S12, contained in the polar face of a putative N-terminal beta-strand, were found to be directly involved in DNA binding, in a manner analogous to RHH proteins . In this respect, TrwA seemed to be a new member of the RHH family . However, secondary structure analyses underscored the existence of a substantial difference in the architecture of the TrwA-oriT complex when compared to the Arc repressor-operator complex. Cell Res, 2004 Oct, 14(5), 400 - 6 The spike protein of severe acute respiratory syndrome (SARS) is cleaved in virus infected Vero-E6 cells; Wu XD et al.; Spike protein is one of the major structural proteins of severe acute respiratory syndrome-coronavirus . It is essential for the interaction of the virons with host cell receptors and subsequent fusion of the viral envelop with host cell membrane to allow infection . Some spike proteins of coronavirus, such as MHV, HCoV-OC43, AIBV and BcoV, are proteolytically cleaved into two subunits, S1 and S2 . In contrast, TGV, FIPV and HCoV-229E are not . Many studies have shown that the cleavage of spike protein seriously affects its function . In order to investigate the maturation and proteolytic processing of the S protein of SARS CoV, we generated S1 and S2 subunit specific antibodies (Abs) as well as N, E and 3CL protein-specific Abs . Our results showed that the antibodies could efficiently and specifically bind to their corresponding proteins from E.coli expressed or lysate of SARS-CoV infected Vero-E6 cells by Western blot analysis . Furthermore, the anti-S1 and S2 Abs were proved to be capable of binding to SARS CoV under electron microscope observation . When S2 Ab was used to perform immune precipitation with lysate of SARS-CoV infected cells, a cleaved S2 fragment was detected with S2-specific mAb by Western blot analysis . The data demonstrated that the cleavage of S protein was observed in the lysate, indicating that proteolytic processing of S protein is present in host cells. J Interferon Cytokine Res, 2004 Sep, 24(9), 553 - 9 Cloning and characterization of Swine interleukin-17, preferentially expressed in the intestines; Katoh S et al.; Interleukin-17 (IL-17), initially reported as CTLA-8, is a proinflammatory cytokine produced mainly by activated T cells . In the present study, the cDNA of a swine IL-17 (PoIL-17) gene was cloned from activated neonatal thymocytes, and the recombinant PoIL-17 (rPoIL-17) was biologically characterized . The complete open reading frame (ORF) of PoIL-17 contains 462-bp coding deduced 153 amino acid residues, with a calculated molecular weight of 17.3 kDa . The amino acid sequence showed 72.9%, 64.9%, 64.7%, 60.1%, and 47.4% similarities with that of human, rat, mouse, Herpesvirus saimiri ORF 13, and chicken, respectively . The six cysteine residues conserved over species including the virus were observed in PoIL-17 . We successfully prepared the recombinant mature form of PoIL-17 and analyzed its biologic activities for swine splenocytes . RT-PCR analysis revealed a marked upregulation of expression of IL-1beta, IL-8, tumor necrosis factor-alpha (TNF-alpha), granulocyte colony-stimulating factor (G-CSF), and monocyte chemotactic protein-1 (MCP-1) mRNA expression in splenocytes treated with 100 ng/ml rPoIL-17 for 3 h . Furthermore, a swine chemokine, alveolar macrophage-derived neutrophil chemotactic factor II (AMCF-II), which was classified into the CXC subfamily was also augmented in mRNA level . This evidence indicates that recombinat PoIL-17 expressed in Escherichia coli was biologically active and exerted similar effects to those of a human (HuIL-17) and murine IL-17 (MuIL-17) . The PoIL-17 mRNA is strongly expressed in the adult heart, skin, and, interestingly, intestinal tissues, including mesenteric lymph nodes but is restricted in neonatal tissues by using real-time quantitative RT-PCR . The gene sequence and biologically active recombinat protein for PoIL-17 will be useful for elucidation of the role of IL-17 in the regulation of intestinal immune responses . Copyright Mary Ann Liebert, Inc. J Interferon Cytokine Res, 2004 Sep, 24(9), 522 - 35 Biochemical analyses of multiple fractions of PKR purified from Escherichia coli; Xu Z et al.; PKR is a cellular protein kinase activated by double-stranded RNA (dsRNA) that phosphorylates eukaryotic initiation factor alpha (eIF2alpha) and inhibits protein translation . Activation of PKR is accompanied by Ser/Thr autophosphorylation on multiple sites . Because PKR negatively regulates cell growth, overexpression and purification of PKR are difficult to achieve . Here, we describe overexpression and purification of recombinant PKR protein from Escherichia coli under native conditions at the milligram level . Affinity, ion exchange, and gel filtration chromatographies revealed multiple fractions of PKR with distinctive biochemical characteristics . During gel filtration, a small amount of PKR was found in a high molecular weight (>300 kDa) fraction that also contained endogenous bacterial RNA . The PKR in this fraction has a constitutive substrate phosphorylation activity . The majority of PKR is found in fractions of lower molecular weight and is free of RNA but is differentially phosphorylated as examined by isoelectric focusing electrophoresis and can be further separated by gradient anion exchange chromatography . PKR eluted with low salt has a lower level of basal autophosphorylation, and its kinase activity can be induced by dsRNA . With an increasing NaCl gradient, the purified PKR exhibits an increased level of autophosphorylation and constitutive kinase activity but reduced dsRNA inducibility . The highest salt eluent of PKR exhibits little dsRNA-induced activation . The inducible activation of high salt eluent PKR by dsRNA can be partially restored by treatment with protein phosphatase 1 . The production of multiple fractions of PKR with different biochemical properties in E . coli suggests that the spectrum of PKR activity and regulation in mammalian cells is likely to be similarly complex . Copyright Mary Ann Liebert, Inc. Biochemistry, 2004 Oct 5, 43(39), 12660 - 6 Nucleotide-dependent isomerization of Escherichia coli RNA polymerase; Lew CM et al.; During promoter engagement, RNA polymerase must change conformation or isomerize to its active form . These data show that high concentrations of nucleotides assist this isomerization . When binding to fork junction DNA probes is monitored, isomerization can occur without the need for the DNA that overlaps the transcription start site . When the start site is present, nucleoside triphosphates cause polymerase to change conformation in a way that drives cross-linking to the +1 position on the template strand . Preincubation of transcription complexes with 2 mM initiating nucleotide can drive formation of heparin-resistant complexes under conditions in which isomerization is limiting . It is proposed that complete polymerase isomerization can require nucleotide binding, which can assist formation of the active site that engages the transcription start site. Biochemistry, 2004 Oct 5, 43(39), 12606 - 13 Sugar binding induced charge translocation in the melibiose permease from Escherichia coli; Meyer-Lipp K et al.; Electrogenic events associated with the activity of the melibiose permease (MelB), a transporter from Escherichia coli, were investigated . Proteoliposomes containing purified MelB were adsorbed to a solid supported lipid membrane, activated by a substrate concentration jump, and transient currents were measured . When the transporter was preincubated with Na(+) at saturating concentrations, a charge translocation in the protein upon melibiose binding could still be observed . This result demonstrates that binding of the uncharged substrate melibiose triggers a charge displacement in the protein . Further analysis showed that the charge displacement is neither related to extra Na(+) binding to the transporter, nor to the displacement of already bound Na(+) within the transporter . The electrogenic melibiose binding process is explained by a conformational change with concomitant displacement of charged amino acid side chains and/or a reorientation of helix dipoles . A kinetic model is suggested, in which Na(+) and melibiose binding are distinct electrogenic processes associated with approximately the same charge displacement . These binding reactions are fast in the presence of the respective cosubstrate (k > 50 s(-1)). Biochemistry, 2004 Oct 5, 43(39), 12585 - 91 Cell-free synthesis of a functional ion channel in the absence of a membrane and in the presence of detergent; Berrier C et al.; We have investigated the possibility of cell-fee synthesis of membrane proteins in the absence of a membrane and in the presence of detergent . We used the bacterial mechanosensitive channel MscL, a homopentamer, as a model protein . A wide range of nonionic or zwitterionic detergents, Triton X-100, Tween 20, Brij 58p, n-dodecyl beta-D-maltoside, and CHAPS, were compatible with cell-free synthesis, while n-octyl beta-D-glucoside and deoxycholate had an inhibitory effect . In vitro synthesis in the presence of Triton X-100 yielded milligram amounts of MscL per milliliter of lysate . Cross-linking experiments showed that the protein was able to oligomerize in detergents . When the purified protein was reconstituted in liposomes and studied by the patch-clamp technique, its activity at the single-molecule level was similar to that of the recombinant protein produced in Escherichia coli . Cell-free synthesis of membrane proteins should prove a valuable tool for the production of membrane proteins whose overexpression in heterologous systems is difficult. Biochemistry, 2004 Oct 5, 43(39), 12539 - 48 Structures of the Escherichia coli PutA proline dehydrogenase domain in complex with competitive inhibitors; Zhang M et al.; Proline dehydrogenase (PRODH) catalyzes the first step of proline catabolism, the flavin-dependent oxidation of proline to Delta(1)-pyrroline-5-carboxylate . Here we present a structure-based study of the PRODH active site of the multifunctional Escherichia coli proline utilization A (PutA) protein using X-ray crystallography, enzyme kinetic measurements, and site-directed mutagenesis . Structures of the PutA PRODH domain complexed with competitive inhibitors acetate (K(i) = 30 mM), L-lactate (K(i) = 1 mM), and L-tetrahydro-2-furoic acid (L-THFA, K(i) = 0.2 mM) have been determined to high-resolution limits of 2.1-2.0 A . The discovery of acetate as a competitive inhibitor suggests that the carboxyl is the minimum functional group recognized by the active site, and the structures show how the enzyme exploits hydrogen-bonding and nonpolar interactions to optimize affinity for the substrate . The PRODH/L-THFA complex is the first structure of PRODH with a five-membered ring proline analogue bound in the active site and thus provides new insights into substrate recognition and the catalytic mechanism . The ring of L-THFA is nearly parallel to the middle ring of the FAD isoalloxazine, with the inhibitor C5 atom 3.3 A from the FAD N5 . This geometry suggests direct hydride transfer as a plausible mechanism . Mutation of conserved active site residue Leu432 to Pro caused a 5-fold decrease in k(cat) and a severe loss in thermostability . These changes are consistent with the location of Leu432 in the hydrophobic core near residues that directly contact FAD . Our results suggest that the molecular basis for increased plasma proline levels in schizophrenic subjects carrying the missense mutation L441P is due to decreased stability of human PRODH2. Biotechnol Bioeng, 2004 Oct 20, 88(2), 189 - 203 Stochastic modeling of the phase-variable pap operon regulation in uropathogenic Escherichia coli; Jarboe LR et al.; Regulation of the pap operon in uropathogenic Escherichia coli is phase variable . This phase variation arises from competition between regulatory proteins at two sites within the regulatory region, GATC(dist) and GATC(prox) . We have used the available literature data to design a stochastic model of the molecular interactions of pap regulation and expression during growth in a non-glucose environment at 37 degrees C . The resulting wild-type model is consistent with reported data . The wild-type model served as a basis for two "in silico" mutant models for investigating the role of key regulatory components, the GATC(dist) binding site and the PapI interaction with Lrp at the GATC(prox) site . Our results show that competition at GATC(dist) is required for phase variation, as previously reported . However, our results suggest that removal of competition at GATC(dist) does not affect initial state dependence . Additionally, the PapI involvement in Lrp translocation from GATC(prox) to GATC(dist) is required for the initial state dependence but not for phase variation . Our results also predict that pap expression is maximized at low growth rates and minimized at high growth rates . These predictions provide a basis for further experimental investigation. Biotechnol Bioeng, 2004 Oct 20, 88(2), 168 - 75 Metabolic engineering of isoprenoid biosynthesis in Arabidopsis for the production of taxadiene, the first committed precursor of Taxol; Besumbes O et al.; Paclitaxel (Taxol) is a widely used anticancer isoprenoid produced by the secondary metabolism of yew (Taxus sp.) trees . However, only limited amounts of Taxol or related metabolites (taxoids) can be obtained from the currently available sources . In this work we have taken the first step toward genetically engineering the biosynthesis of taxoids in angiosperms . The first committed step in Taxol biosynthesis is the production of taxadiene from geranylgeranyl diphosphate (GGPP), catalyzed by the plastid-localized enzyme taxadiene synthase (TXS) . A recombinant T . baccata TXS lacking the putative plastid targeting peptide and fused to a C-terminal histidine (His) tag was shown to be enzymatically active in Escherichia coli . Constitutive production of the full-length His-tagged enzyme in Arabidopsis thaliana plants led to the accumulation of taxadiene and concomitant growth retardation and decreased levels of photosynthetic pigment in transgenic plants . Although these phenotypes may derive from a toxic effect of taxadiene, the lower accumulation of endogenous plastid isoprenoid products such as carotenoids and chlorophylls in transgenic plants also suggests that the constitutive production of an active TXS enzyme might alter the balance of the GGPP pool . Induction of transgene expression using a glucocorticoid-mediated system consistently resulted in a more efficient recruitment of GGPP for the production of taxadiene, which reached levels 30-fold higher than those in plants constitutively expressing the transgene . This accomplishment illustrates the possibility of engineering the production of taxoids and other GGPP-derived isoprenoids in crop plants despite the constraints associated with limited knowledge with regard to regulation of GGPP availability. Arch Virol, 2005 Jan, 150(1), 21 - 36 Epub 2004 Sep 24. Cross-reactivity among sapovirus recombinant capsid proteins; Hansman GS et al.; Sapovirus (SaV), a member of the genus Sapovirus in the family Caliciviridae, is an agent of human and porcine gastroenteritis . SaV strains are divided into five genogroups (GI-GV) based on their capsid (VP1) sequences . Human SaV strains are noncultivable, but expression of the recombinant capsid protein (rVP1) in a baculovirus expression system results in the self-assembly of virus-like particles (VLPs) that are morphologically similar to native SaV . In this study, rVP1 constructs of SaV GI, GII, and GV strains were expressed in a baculovirus expression system . The structures of the GI, GII, and GV VLPs, with diameters of 41-48 nm, were morphologically similar to those of native SaV . However a fraction of GV VLPs were smaller, with diameters of 26-31 nm and spikes on the outline . This is the first report of GII and GV VLP formation and the first identification of small VLPs . To examine the cross-reactivities among GI, GII, and GV rVP1, hyperimmune rabbit antisera were raised against Escherichia coli-expressed GI, GII, and GV N- and C-terminal VP1 . Western blotting showed the GI antisera cross-reacted with GV rVP1 but not GII rVP1; GII antisera cross-reacted weakly with GI rVP1 but did not cross-react with GV rVP1; and GV antisera reacted only with GV rVP1 . Also, hyperimmune rabbit and guinea pig antisera raised against purified GI VLPs were used to examine the cross-reactivities among GI, GII, and GV VLPs by an antigen enzyme-linked immunosorbent assay (ELISA) . The ELISA showed that the GI VLPs were antigenically distinct from GII and GV VLPs. Acta Biochim Pol, 2004, 51(3), 857 - 60 Role of rpoS in the regulation of glyoxalase III in Escherichia coli; Benov L et al.; Methylglyoxal is an endogenous electrophile produced in Escherichia coli by the enzyme methylglyoxal synthase to limit the accumulation of phosphorylated sugars . In enteric bacteria methylglyoxal is detoxified by the glutathione-dependent glyoxalase I/II system, by glyoxalase III, and by aldehyde reductase and alcohol dehydrogenase . Here we demonstrate that glyoxalase III is a stationary-phase enzyme . Its activity reached a maximum at the entry into the stationary phase and remained high for at least 20 h . An rpoS- mutant displayed normal glyoxalase I and II activities but was unable to induce glyoxalase III in stationary phase . It thus appears that glyoxalase III is regulated by rpoS and might be important for survival of non-growing E . coli cultures. Acta Biochim Pol, 2004, 51(3), 723 - 32 CEA-negative glioblastoma and melanoma cells are sensitive to cytosine deaminase/5-fluorocytosine therapy directed by the carcinoembryonic antigen promoter; Dabrowska A et al.; Recent studies have suggested that carcinoembryonic antigen (CEA)-promoter sequences are active only in CEA-positive cells, filing in the criteria for tumor specific targeting of suicide genes . However, the present study on gene therapy of colon cancer and cell-specificity of CEA promoter, provide evidence that CEA-positive and CEA-negative cells transfected with E . coli cytosine deaminase (CD) gene under the control of CEA promotor sequence are sensitive to enzyme/pro-drug therapy with 5-fluorocytosine (5-FC) . Individual clones derived from the CEA-negative cell lines: melanoma Hs294T and glioblastoma T98G after transfection with CD differed profoundly in their sensitivity to 5-FC . The IC50 values for several clones of the CEA-negative cells were almost the same as for CEA-positive colon cancer cells . Such 5-FC-sensitive clones derived from the population of CEA-negative cells, present even in small number, because of the very effective bystender effect of this enzyme/pro-drug system can cause severe problems during therapy by efficiently killing surrounding normal cells . Safety is the major issue in gene therapy . Our data suggest that the safety of gene-directed enzyme pro-drug therapy (GDEPT) with CEA promoter driven expression of therapeutic genes is not so obvious as it has originally been claimed. Acta Biochim Pol, 2004, 51(3), 683 - 92 Spontaneous mutagenesis in exponentially growing and stationary-phase, umuDC-proficient and -deficient, Escherichia coli dnaQ49; Nowosielska A et al.; Spontaneous mutations arise not only in exponentially growing bacteria but also in non-dividing or slowly dividing stationary-phase cells . In the latter case mutations are called adaptive or stationary-phase mutations . High spontaneous mutability has been observed in temperature sensitive Escherichia coli dnaQ49 strain deficient in 3'-->5' proofreading activity assured by the e subunit of the main replicative polymerase, Pol III . The aim of this study was to evaluate the effects of the dnaQ49 mutation and deletion of the umuDC operon encoding polymerase V (Pol V) on spontaneous mutagenesis in growing and stationary-phase E . coli cells . Using the argE3(OC) -->Arg+ reversion system in the AB1157 strain, we found that the level of growth-dependent and stationary-phase Arg+ revertants was significantly increased in the dnaQ49 mutant at the non-permissive temperature of 37 degrees C . At this temperature, in contrast to cultures grown at 28 degrees C, SOS functions were dramatically increased . Deletion of the umuDC operon in the dnaQ49 strain led to a 10-fold decrease in the level of Arg+ revertants in cultures grown at 37 degrees C and only to a 2-fold decrease in cultures grown at 28 degrees C . Furthermore, in stationary-phase cultures Pol V influenced spontaneous mutagenesis to a much lesser extent than in growing cultures . Our results indicate that the level of Pol III desintegration, dependent on the temperature of incubation, is more critical for spontaneous mutagenesis in stationary-phase dnaQ49 cells than the presence or absence of Pol V. Nat Cell Biol, 2004 Oct, 6(10), 991 - 6 Epub 2004 Sep 26. Recruitment of Xenopus Scc2 and cohesin to chromatin requires the pre-replication complex; Takahashi TS et al.; Cohesin is a multi-subunit, ring-shaped protein complex that holds sister chromatids together from the time of their synthesis in S phase until they are segregated in anaphase . In yeast, the loading of cohesin onto chromosomes requires the Scc2 protein . In vertebrates, cohesins first bind to chromosomes as cells exit mitosis, but the mechanism is unknown . Concurrent with cohesin binding, pre-replication complexes (pre-RCs) are assembled at origins of DNA replication through the sequential loading of the initiation factors ORC, Cdc6, Cdt1 and MCM2-7 (the 'licensing' reaction) . In S phase, the protein kinase Cdk2 activates pre-RCs, causing origin unwinding and DNA replication . Here, we use Xenopus egg extracts to show that the recruitment of cohesins to chromosomes requires fully licensed chromatin and is dependent on ORC, Cdc6, Cdt1 and MCM2-7, but is independent of Cdk2 . We further show that Xenopus Scc2 is required for cohesin loading and that binding of XScc2 to chromatin is MCM2-7 dependent . Our results define a novel pre-RC-dependent pathway for cohesin recruitment to chromosomes in a vertebrate model system. Nat Genet, 2004 Oct, 36(10), 1056 - 8 Epub 2004 Sep 26. Metabolic gene-deletion strains of Escherichia coli evolve to computationally predicted growth phenotypes; Fong SS et al.; Genome-scale metabolic models have a promising ability to describe cellular phenotypes accurately . Here we show that strains of Escherichia coli carrying a deletion of a single metabolic gene increase their growth rates (by 87% on average) during adaptive evolution and that the endpoint growth rates can be predicted computationally in 39 of 50 (78%) strains tested . These results show that computational models can be used to predict the eventual effects of genetic modifications. Proc Natl Acad Sci U S A, 2004 Oct 12, 101(41), 14689 - 94 Epub 2004 Sep 24. Local graph alignment and motif search in biological networks; Berg J et al.; Interaction networks are of central importance in postgenomic molecular biology, with increasing amounts of data becoming available by high-throughput methods . Examples are gene regulatory networks or protein interaction maps . The main challenge in the analysis of these data is to read off biological functions from the topology of the network . Topological motifs, i.e., patterns occurring repeatedly at different positions in the network, have recently been identified as basic modules of molecular information processing . In this article, we discuss motifs derived from families of mutually similar but not necessarily identical patterns . We establish a statistical model for the occurrence of such motifs, from which we derive a scoring function for their statistical significance . Based on this scoring function, we develop a search algorithm for topological motifs called graph alignment, a procedure with some analogies to sequence alignment . The algorithm is applied to the gene regulation network of Escherichia coli. J Exp Bot, 2004 Dec, 55(408), 2483 - 93 Epub 2004 Sep 24. A plant type 2 metallothionein (MT) from cork tissue responds to oxidative stress; Mir G et al.; Expression of plant metallothionein genes has been reported in a variety of senescing tissues, such as leaves and stems, ripening fruits, and wounded tissues, and has been proposed to function in both metal chaperoning and scavenging of reactive oxygen species . In this work, it is shown that MT is also associated with suberization, after identifying a gene actively transcribed in Quercus suber cork cells as a novel MT . This cDNA, isolated from a phellem cDNA library, encodes a MT that belongs to type 2 plant MTs (QsMT) . Expression of the QsMT cDNA in E . coli grown in media supplemented with Zn, Cd, or Cu has yielded recombinant QsMT . Characterization of the respective metal aggregates agrees well with a copper-related biological role, consistent with the capacity of QsMT to restore copper tolerance to a MT-deficient, copper-sensitive yeast mutant . Furthermore, in situ hybridization results demonstrate that RNA expression of QsMT is mainly observed under conditions related to oxidative stress, either endogenous, as found in cork or in actively proliferating tissues, or exogenous, for example, in response to H(2)O(2) or paraquat treatments . The putative role of QsMT in oxidative stress, both as a free radical scavenger via its sulphydryl groups or as a copper chelator is discussed. J Biol Chem, 2004 Dec 3, 279(49), 51442 - 50 Epub 2004 Dec 3. HucR, a novel uric acid-responsive member of the MarR family of transcriptional regulators from Deinococcus radiodurans; Wilkinson SP et al.; The MarR family of transcriptional regulators comprises a subset of winged helix DNA-binding proteins and includes numerous members that function in environmental surveillance of aromatic compounds . We describe the characterization of HucR, a novel MarR homolog from Deinococcus radiodurans that demonstrates phenolic sensing capabilities . HucR binds as a homodimer to a single site within its promoter/operator region with Kd = 0.29 +/- 0.02 nM . The HucR binding site contains a pseudopalindromic sequence, composed of 8-bp half-sites separated by 2 bp . The location of the HucR binding site in the intergenic region between hucR and a putative uricase suggests a mechanism of simultaneous co-repression of these two genes . The substrate of uricase, uric acid, is an efficient antagonist of DNA binding, reducing HucR-DNA complex formation to 50% at 0.26 mM ligand, compared with 5.2 and 46 mM for the aromatic compounds salicylate and acetylsalicylate, respectively . Enhanced levels in vivo of hucR and uricase transcript and increased uricase activity under conditions of excess uric acid further indicate a novel regulatory mechanism of aromatic catabolism in D . radiodurans . Since uric acid is a scavenger of reactive oxygen species, we hypothesize that HucR is a participant in the intrinsic resistance of D . radiodurans to high levels of oxidative stress. J Biol Chem, 2004 Dec 3, 279(49), 50994 - 1001 Epub 2004 Dec 3. Characterization of mammalian sulfiredoxin and its reactivation of hyperoxidized peroxiredoxin through reduction of cysteine sulfinic acid in the active site to cysteine; Chang TS et al.; Peroxiredoxins (Prxs) are a family of peroxidases that reduce hydroperoxides . The cysteine residue in the active site of certain eukaryotic Prx enzymes undergoes reversible oxidation to sulfinic acid (Cys-SO2H) during catalysis, and sulfiredoxin (Srx) has been identified as responsible for reversal of the resulting enzyme inactivation in yeast . We have now characterized mammalian orthologs of yeast Srx with an assay based on monitoring of the reduction of sulfinic Prx by immunoblot analysis with antibodies specific for the sulfinic state . Sulfinic reduction by mammalian Srx was found to be a slow process (kcat = 0.18/min) that requires ATP hydrolysis . ATP could be efficiently replaced by GTP, dATP, or dGTP but not by CTP, UTP, dCTP, or dTTP . Both glutathione and thioredoxin are potential physiological electron donors for the Srx reaction, given that their Km values (1.8 mM and 1.2 microM, respectively) are in the range of their intracellular concentrations, and the Vmax values obtained with the two reductants were similar . Although its pKa is relatively low (approximately 7.3), the active site cysteine of Srx remained reduced even when the active site cysteine of most Prx molecules became oxidized . Finally, depletion of human Srx by RNA interference suggested that Srx is largely responsible for reduction of the Cys-SO2H of Prx in A549 human cells. J Biol Chem, 2004 Dec 3, 279(49), 51568 - 73 Epub 2004 Dec 3. Prereplicative purine methylation and postreplicative demethylation in each DNA duplication of the Escherichia coli replication cycle; Maas R; Escherichia coli plasmid DNA activated for initiation of duplication is in a stable low linking number supercoiled conformation . Low linking number DNA is methylated at the internal purines of a frequent 5'-Pyr-Pyr-Pur-Pur tetramer with a 5'-Pyr-Pur-3' axis of symmetry and is cut at the axis of symmetry by pneumococcal restriction enzyme DpnI when methylated in both strands . Purine methylation is of adenine in one strand and guanine in the other . Methylation of one of the two purines is removed during the cell cycle, presumably before the reverse shift to the B-supercoiled conformation . The topological transition was reconstituted in vitro only with DNA unmethylated at purines . Methylation-restriction analyses coupled with the chemical properties of low-linking number DNA and B-DNA respectively, suggest that removal of guanine methylation is essential for the low-linking number to B-DNA transition and hence for the deactivation of replication . Demethylation of methylguanine could explain the presence in E . coli of the two-member inducible operon known as ada . Characteristics of ada suggest a cascade of chemical DNA modifications that reverse prereplicative guanine methylation . Guanine demethylation could provide a model for the pivotal role played by de novo methylation in replication and for the essential role of "repair" enzyme ExoIII in demethylation leading to the reversal of replicative DNA activation and other processes that affect DNA function. J Biol Chem, 2004 Dec 3, 279(49), 51534 - 40 Epub 2004 Dec 3. Structural basis of biopterin-induced inhibition of GTP cyclohydrolase I by GFRP, its feedback regulatory protein; Maita N et al.; GTP cyclohydrolase I (GTPCHI) is the rate-limiting enzyme involved in the biosynthesis of tetrahydrobiopterin, a key cofactor necessary for nitric oxide synthase and for the hydroxylases that are involved in the production of catecholamines and serotonin . In animals, the GTPCHI feedback regulatory protein (GFRP) binds GTPCHI to mediate feed-forward activation of GTPCHI activity in the presence of phenylalanine, whereas it induces feedback inhibition of enzyme activity in the presence of biopterin . Here, we have reported the crystal structure of the biopterin-induced inhibitory complex of GTPCHI and GFRP and compared it with the previously reported phenylalanine-induced stimulatory complex . The structure reveals five biopterin molecules located at each interface between GTPCHI and GFRP . Induced fitting structural changes by the biopterin binding expand large conformational changes in GTPCHI peptide segments forming the active site, resulting in inhibition of the activity . By locating 3,4-dihydroxy-phenylalanine-responsive dystonia mutations in the complex structure, we found mutations that may possibly disturb the GFRP-mediated regulation of GTPCHI. Di Yi Jun Yi Da Xue Xue Bao, 2004 Sep, 24(9), 1033 - 6 {An initial examination of the spermatozoal gene expression profile}; Mao XM et al.; OBJECTIVE: To collect the normal spermatozoal gene expression sequence tags with the restriction display technique for constructing a microarray to understand spermatozoal gene expression profiles . METHODS: The total RNA extracted from normal human spermatozoa were reversely transcribed into cDNAs, which were digested by Sau3AI and linked to universal adapters (adapter 1) at both ends . According to the sequence of the adapter, a pair of primers (universal primers 1) was designed, followed by PCR with primers 1 and the PCR products were transferred into E.coli . The positive clones were collected after identification to serve as the probes for constructing the gene expression microarray of spermatozoa by printing those probes on the slides . The accomplished microarrays were examined by Cy3-labeled normal spermatozoal samples . RESULTS: Altogether 1 859 probes were collected, from which 368 were picked out randomly for constructing the microarray . CONCLUSIONS: Human spermatozoa contain a rich repertoire of RNAs, and the probes we prepared possess good incredibility and speciality. Biochem J . 2004 Sep 24; Pt {Epub ahead of print} Substrate and product specificities of cis-type undecaprenyl pyrophosphate synthase; Chen AP et al.; Undecaprenyl pyrophosphate synthase (UPPs) catalyzes consecutive condensation reactions of farnesyl pyrophosphate (FPP) with eight isopentenyl pyrophosphate (IPP) to generate C 55 undecaprenyl pyrophosphate (UPP), which serves as a lipid carrier for bacterial peptidoglycan biosynthesis . We reported the co-crystal structure of E . coli UPPs in complex with FPP . Its phosphate head-group is bound to the positively-charged Arg residues and the hydrocarbon moiety interacts with hydrophobic amino acids including Leu85, Leu88, and Phe89, located on the alpha3 helix of UPPs . We now show the mono-phosphate analogue of FPP binds UPPs with a 8-fold lower affinity (K d=4.4 muM) compared with the pyrophosphate analogue, a result of a larger dissociation rate constant (k off=192 s -1) . Farnesol (1 mM) lacking the pyrophosphate does not inhibit the UPPs reaction . Geranylgeranyl pyrophosphate (GGPP) containing a larger C 20 hydrocarbon tail is an equally good substrate (K m=0.3 muM and k cat=2.1 s -1) compared with FPP . The shorter C 10 GPP displays a 90-fold larger K m value (36.0+/-0.1 muM) but similar k cat value (1.7+/-0.1 s -1) compared with FPP . Replacement of Leu85, Leu88, or Phe89 with Ala increases FPP and GGPP K m values by the same amount, indicating that these amino acids are important for substrate binding, but do not determine substrate specificity . With GGPP as a substrate, UPPs still catalyzes eight IPP condensation reactions to synthesize C 60 product . Computer-modeling suggests that the upper portion of the active site tunnel, where cis-double bonds of the product reside, may be critical in determining the final product chain length. Phys Rev Lett . 2004 Aug 27;93(9):098104 . Epub 2004 Aug 25. Coarse-grained model of entropic allostery; Hawkins RJ et al.; Many signaling functions in molecular biology require proteins to bind to substrates such as DNA in response to environmental signals such as the simultaneous binding to a small molecule . Examples are repressor proteins which may transmit information via a conformational change in response to the ligand binding . An alternative entropic mechanism of "allostery" suggests that the inducer ligand changes the intramolecular vibrational entropy, not just the mean static structure . We present a quantitative, coarse-grained model of entropic allostery, which suggests design rules for internal cohesive potentials in proteins employing this effect . It also addresses the issue of how the signal information to bind or unbind is transmitted through the protein . The model may be applicable to a wide range of repressors and also to signaling in trans-membrane proteins. J Anim Sci, 2004 Sep, 82(9), 2657 - 66 The efficacy of an Escherichia coli-derived phytase preparation; Adeola O et al.; Five experiments were conducted to evaluate the effect of an Escherichia coli-derived phytase on phytate-P use and growth performance by young pigs . The first experiment involved time course, pH dependence, and phytase activity studies to investigate the in vitro release of P from corn, soybean meal, and an inorganic P-unsupplemented corn-soybean meal negative control diet . In Exp . 2, which was designed to determine the efficacy of the E . coli-derived vs . fungal phytase-added diets at 0, 250, 500, 750, 1,000, or 1,250 FTU/kg (as-fed basis; one phytase unit or FTU is defined as the quantity of enzyme required to liberate 1 micromol of inorganic P/min, at pH 5.5, from an excess of 15 microM sodium phytate at 37 approximately C) and a positive control diet, eight individually penned 10-kg pigs per diet (12 diets, 96 pigs) were used in a 28-d growth study . The third experiment was a 10-d nutrient balance study involving six 13-kg pigs per diet (four diets, 24 pigs) in individual metabolism crates . In Exp . 4, eight pens (four pigs per pen) of 19-kg pigs per treatment were used in a 42-d growth performance study to examine the effect of adding the E . coli-derived phytase to corn-soybean diets at 0, 500, or 1,000 FTU/kg (as-fed basis) and a positive control (four diets, 128 pigs) . In Exp . 5, six 19-kg pigs per treatment were used in a 10-d nutrient balance study to investigate the effects of the E . coli-derived phytase added to diets at 0, 250, 500, 750, or 1,000 FTU/kg (as-fed basis) and a positive control diet (six diets, 36 pigs) . The in vitro study showed that the E . coli-derived phytase has an optimal activity and pH range of 2 to 4.5 . Inorganic phosphate release was greatest for soybean meal, least for corn, and intermediate for the negative control diet . Dietary supplementation with graded amounts of E . coli-derived phytase resulted in linear increases (P < 0.05) in weight gain, feed efficiency, and plasma Ca and P concentrations in 10-kg pigs in Exp . 2 . Phytase also increased P digestibility and retention in the 13-kg pigs in Exp . 3 . In Exp . 4, dietary supplementation with E . coli-derived phytase resulted in linear increases (P < 0.05) in weight gain and feed efficiency of 19-kg pigs . Supplementation of the diets of 19-kg pigs with the E . coli-derived phytase also improved Ca and P digestibility and retention in Exp . 5 . In the current study, the new E . coli-derived phytase was efficacious in hydrolyzing phyt