Gene, 1995 Dec 29, 167(1-2), 69 - 74 The creation of a camptothecin-sensitive Escherichia coli based on the expression of the human topoisomerase I; Taylor ST et al.; The synthesis of the human topoisomerase I (Topo I) in Escherichia coli (Ec) results in phenotypes consistent with the appearance of a DNA-relaxing activity . High-level expression was problematic and recloning the gene in a reduced-copy-number plasmid under more stringently regulated control produced a stable plasmid . An Ec strain with an inducible sensitivity to the eukaryotic Topo I poison, camptothecin (CPT), was constructed by introducing a mutation (imp4312) known to enhance the permeability of Ec to a wide variety of compounds . We are able to infer that CPT sensitivity in Ec involves DNA damage by noting elevated sensitivity in a repair-deficient recA strain and by observing the induction of a sulA::lac fusion following exposure to the drug. Gene, 1995 Dec 29, 167(1-2), 63 - 8 Rapid purification of recombinant proteins fused to chicken avidin; Airenne KJ et al.; A novel expression vector (pAVEX16C) has been constructed that directs the synthesis of desired polypeptides as fusions with the C terminus of chicken egg-white avidin (Avd) . With this and a commercial GST gene (encoding glutathione S-transferase) fusion vector (pGEX-3X, Pharmacia), we produced Avd as fusions C- and N-terminally linked to GST in Escherichia coli . By using the Avd tail and a simple affinity purification protocol, including biotin-agarose, we were able to obtain 1-2 micrograms/ml of highly purified Avd::GST and GST::Avd from crude bacterial lysates . The produced proteins were, to a great extent, in soluble fraction when the cells were grown at 22 degrees C and disrupted with a detergent, N-laurylsarcosine . The fusion proteins could also be affinity-purified with the GST tail using glutathione-Sepharose 4B, but the yield of GST::Avd was significantly lower than when using the Avd tail . Our results therefore indicate that it is possible to produce, in E . coli, biologically active fusion proteins consisting of Avd C- or N-terminally linked with the desired protein which then can easily be purified by a simple affinity chromatography procedure. Gene, 1995 Dec 29, 167(1-2), 53 - 7 An improved TnMax mini-transposon system suitable for sequencing, shuttle mutagenesis and gene fusions; Kahrs AF et al.; A new collection of mini-transposons (mini-Tn) of the previously described TnMax series {Haas et al, Gene 130 (1993a) 23-31} has been constructed . The transposons (Tn) bear genes conferring resistance to either chloramphenicol (Cm) or kanamycin (Km) . Each member of the new series (TnMax5-TnMax11) contains the general M13 forward (M13-FP) and reverse (M13-RP1) sequencing primers close to the inverted repeats (IR), facilitating the rapid and convenient determination of the DNA sequences flanking the transposon insertion site . Furthermore, the mini-Tn possess the infrequently occurring NotI sites, allowing the localization of genes on macro-restriction maps of bacterial species . Some derivatives contain promoterless trp-lacZ (TnMax11), xylE (TnMax10), phoA (TnMax6) or blaM (TnMax7, TnMax9) genes next to the IR, suitable for the generation of in vivo gene- and operon fusions to study gene regulation, protein export, or to determine the topology of proteins in bacterial membranes . A set of conjugative minimal plasmid vectors (pMin1, pMin2) are used to select for TnMax insertions into the cloned insert, rather than the vector sequences . Due to the small size of the mini-Tn, and a simple and efficient mutagenesis procedure, the TnMax system is a useful tool for targeting and sequencing of cloned genes in Escherichia coli, and especially for shuttle mutagenesis of bacterial species which cannot be targeted by direct transposition. Gene, 1995 Dec 29, 167(1-2), 227 - 31 The Caenorhabditis elegans rop-1 gene encodes the homologue of the human 60-kDa Ro autoantigen; Labbe JC et al.; As a first step toward establishing a genetic system for the elucidation of the cellular role(s) of the Ro ribonucleoproteins (RoRNP), we have cloned the gene encoding the homologue of the human 60-kDa Ro protein (Ro60) in Caenorhabditis elegans (Ce) . This Ce gene is present as a single copy and contains a 643-codon open reading frame interrupted by three introns . The encoded protein, Rop1p, shares 40% identity and 63% overall similarity with both the human and amphibian Ro60 . Recombinant protein has been produced in Escherichia coli and used to elicit anti-Rop1p antibodies . Immunological analysis indicated that the Ro60 epitopes have been poorly conserved . Gene-fusion expression studies in transgenic nematodes will provide a new avenue of research to shed light on the function of these particles. Gene, 1995 Dec 29, 167(1-2), 17 - 24 Characterization of two members (ACS1 and ACS3) of the 1-aminocyclopropane-1-carboxylate synthase gene family of Arabidopsis thaliana; Liang X et al.; The nucleotide sequences of two highly homologous 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS; EC 4.4.1.14)-encoding genes, ACS1 and ACS3, of Arabidopsis thaliana (At) have been determined . The sequence analysis shows that ACS3 is a pseudogene representing a truncated version of ACS1 . The missing region of ACS3 corresponding to the fourth exon of ACS1 has been shown by Southern analysis to be absent in the At genome . The chromosomal locations of the five members of the At ACS multigene family have been determined . The results show that each family member resides on a different chromosome . This observation suggests that the ACS3 pseudogene originated by a partial inter-chromosomal gene duplication . The ACS1 polypeptide contains all the conserved and characteristic domains found in the ACC synthase isoenzymes from various plant species, but is unable to express ACS activity in Escherichia coli and yeast . The predicted amino-acid sequence of ACS1 is missing the highly conserved tripeptide, Thr-Asn-Pro (TNP), between Ile204 and Ser205 . Introduction of TNP into ACS1 restores the ACS activity, whereas its removal from the enzymatically active ACS2 results in a loss of activity . The results suggest that TNP is crucial for expression of ACS activity in E . coli. Gene, 1995 Dec 29, 167(1-2), 147 - 9 A putative SOS repair gene (dinF-like) in a hyperthermophilic archaeon; Bouyoub A et al.; The hyperthermophilic archaeon, Pyrococcus (strain IFREMER AL585), contains an ORF that encodes a polypeptide with a high similarity to the Escherichia coli dinF (DNA damage-inducible) gene product . The conservation of this protein between Archaea and Bacteria suggests that a SOS repair system might operate in Archaea. Gene, 1995 Dec 29, 167(1-2), 133 - 6 Identification of alcA, a Bordetella bronchiseptica gene necessary for alcaligin production; Giardina PC et al.; The alcA gene, essential for the production of the dihydroxamate siderophore, alcaligin, by Bordetella bronchiseptica, was cloned and sequenced . The alcA gene was identified on a 4.7-kb EcoRI genomic fragment adjacent to a Tn5lac transposon insertion that inactivated alcaligin production in strain MBORD846 . Analysis of the alcA nucleotide sequence revealed a putative Fur-binding site, suggesting that expression of this gene is repressed by iron . The deduced amino-acid sequence of this open reading frame had significant homology with the Escherichia coli iucD gene product, an enzyme required for biosynthesis of the dihydroxamate siderophore aerobactin. Gene, 1995 Dec 29, 167(1-2), 127 - 32 Cloning, expression and sequence analysis of the genes encoding the heterodimeric methylmalonyl-CoA mutase of Porphyromonas gingivalis W50; Jackson CA et al.; Two genes that encode methylmalonyl-CoA mutase (MCM) have been characterised in Porphyromonas gingivalis W50 (Pg) . The genes, designated mcmA and mcmB are transcribed in an operon and encode the MCM small subunit (SS, 68,626 Da) and the MCM large subunit (LS, 78,703 Da), respectively . A recombinant Escherichia coli (Ec) clone harbouring the Pg mcmA and mcmB genes expressed MCM activity 280-times higher than that of the Ec control . The C terminus of the MCM LS has sequence homology to domains of a variety of enzymes that consume or produce methylmalonyl-CoA, suggesting that the MCM LS C-terminal domain is involved in substrate binding . The MCM LS C-terminal region also exhibits homology to other enzymes that have cobalamin-containing cofactors . It is likely, therefore, that the C terminus of the MCM LS is an important MCM domain involved in both substrate and cofactor binding. Cell, 1995 Dec 29, 83(7), 1171 - 81 SecA membrane cycling at SecYEG is driven by distinct ATP binding and hydrolysis events and is regulated by SecD and SecF; Economou A et al.; The SecA subunit of E . coli preprotein translocase promotes protein secretion during cycles of membrane insertion and deinsertion at SecYEG . This process is regulated both by nucleotide binding and hydrolysis and by the SecD and SecF proteins . In the presence of associated preprotein, the energy of ATP binding at nucleotide-binding domain 1 (NBD1) drives membrane insertion of a 30 kDa domain of SecA, while deinsertion of SecA requires the hydrolysis of this ATP . SecD and SecF stabilize the inserted state of SecA . ATP binding at NBD2, though needed for preprotein translocation, is not needed for SecA insertion or deinsertion. J Biol Chem, 1995 Dec 29, 270(52), 31397 - 404 An interaction between the Escherichia coli RecF and RecR proteins dependent on ATP and double-stranded DNA; Webb BL et al.; The DNA binding and ATPase activities of RecF protein are modulated by RecR protein . Stoichiometric amounts of RecF protein bind to double-stranded (ds) DNA (about 1 RecF monomer/4-6 base pairs) in the presence of adenosine 5'-O-(3-thio)triphosphate (ATP gamma S), forming a homogeneous protein coating on the DNA . Little or no cooperativity is evident in the binding process . In the presence of ATP, RecF binding to dsDNA is much weaker, and no RecF protein coating forms . Instead, small numbers of RecF protomers are interspersed randomly along the DNA . RecR protein does not bind appreciably to the dsDNA under these same conditions . However, a protein coating, similar to that which was observed with RecF protein alone in the presence of ATP gamma S, was produced when both RecF and RecR proteins were incubated with dsDNA in the presence of ATP . An interaction between RecF and RecR enables both proteins to bind tightly to the dsDNA in an approximately 1:1 molar ratio . We also report a weak ATP hydrolytic activity of RecF which is stimulated by RecR. J Biol Chem, 1995 Dec 29, 270(52), 31345 - 52 Cloning and expression of a cDNA encoding the beta-subunit (30-kDa subunit) of bovine brain platelet-activating factor acetylhydrolase; Hattori M et al.; Bovine brain platelet-activating factor (PAF) acetylhydrolase isoform Ib is a heterotrimeric enzyme . Its gamma-subunit (which, formerly, we called the 29-kDa subunit) acts as a catalytic subunit, whereas the alpha-subunit (45 kDa) is the bovine homolog of the product of human LIS-1, the causative gene of Miller-Dieker lissencephaly, indicating that this intracellular PAF acetylhydrolase plays a key role in brain development . In the current study, we cloned the cDNA for the beta-subunit (30 kDa) of bovine brain PAF acetylhydrolase Ib . The predicted 229-amino acid sequence was homologous (63.2% identity) to that of the gamma-subunit, especially (86% identity) in the catalytic and PAF receptor homologous domains . The recombinant beta-protein produced in Escherichia coli showed significant PAF acetylhydrolase activity . A mutant protein, in which Ser48, which corresponds to the active serine residue of the gamma-subunit, was replaced with cysteine showed no enzymatic activity, suggesting Ser48 is the active serine residue . Although the beta- and gamma-subunits form a heterocomplex in the native enzyme, both recombinant beta- and gamma-proteins exist as a homodimer . The purified recombinant beta-protein was labeled readily with {1,3-H}diisopropyl fluorophosphate, whereas the beta-subunit in the native complex was only labeled with higher concentrations of {1,3-3H}diisopropyl fluorophosphate to a lesser extent than the gamma-subunit . Combined with our previous data, the present study demonstrated that bovine brain PAF acetylhydrolase Ib is a unique enzyme possessing two catalytic subunits and another, possibly regulatory, subunit. J Biol Chem, 1995 Dec 29, 270(52), 31303 - 9 Presence of N-unsubstituted glucosamine units in native heparan sulfate revealed by a monoclonal antibody; van den Born J et al.; Immunohistochemical application of antibodies against heparan sulfate proteoglycan core protein and heparitinase-digested heparan sulfate stubs showed the presence of heparan sulfate proteoglycan in all basement membranes of the rat kidney . However, a monoclonal antibody (JM-403) against native heparan sulfate (van den Born, J., van den Heuvel, L . P . W . J., Bakker, M . A . H., Veerkamp, J . H., Assmann, K . J . M., and Berden, J . H . M . (1992) Kidney Int . 41, 115-123) largely failed to stain tubular basement membranes, suggesting the presence of heparan sulfate chains lacking the specific JM-403 epitope . Heparan sulfate preparations from various sources differed markedly with regard to JM-403 binding, as demonstrated by liquid phase inhibition in enzyme-linked immunosorbent assay, the interaction decreasing with increasing sulfate contents of the polysaccharide . Mapping of the JM-403 epitope indicated that it was dominated by one or more N-unsubstituted glucosamine unit(s), since treatments that destroyed or altered the structure of such units in heparan sulfate preparations (cleavage at N-unsubstituted glucosamine units with HNO2 at pH 3.9 and N-acetylation with acetic anhydride, respectively), abolished antibody binding . Conversely, immunoreactivity could be induced in a (D-glucuronyl-1,4-N-acetyl-D-glucosaminyl-1,4) polysaccharide by the generation of N-unsubstituted glucosamine N-unsubstituted glucosamine in a JM-403-binding heparan sulfate (preparation HS-II from human aorta) was demonstrated by an approximately 3-fold reduction in molecular size following HNO2 (pH 3.9) treatment . Further characterization of the epitope recognized by JM-403, based on enzyme-linked immunosorbent assay inhibition tests with chemically/enzymatically modified polysaccharides, indicated that one or more N-sulfated glucosamine units are invariable present, whereas L-iduronic acid and O-sulfate residues appear to inhibit JM-403 reactivity . It is concluded that the epitope contains one or more N-unsubstituted glucosamine and D-glucuronic acid units and is located in a region of the heparan sulfate chain composed of mixed N-sulfated and N-acetylated disaccharide units. J Biol Chem, 1995 Dec 29, 270(52), 31262 - 8 Molecular characterization of Golgin-245, a novel Golgi complex protein containing a granin signature; Fritzler MJ et al.; The serum from a Sjogren's syndrome patient with anti-Golgi antibodies was used as a probe to isolate a 4.6-kilobase pair cDNA insert from a HeLa cDNA library . Expression of the cDNA in Escherichia coli and the in vitro translation products of the cDNA yielded a recombinant protein that migrated in SDS-polyacrylamide gel electrophoresis at 180 kDa . This protein was immuno-precipitated by the human anti-Golgi serum and by immune rabbit serum but not by normal human serum or preimmune rabbit serum . Western blot analysis showed that the prototype human and immune rabbit sera recognized a 245-kDa protein, suggesting that the isolated clone contained a partial cDNA . The 5'-upstream sequence obtained by the rapid amplification of cDNA ends methodology using human placental cDNA and the combined HeLa cDNA contained 6965 base pairs and combined HeLa cDNA contained 6965 base pairs and encoded a protein of 245 kDa and, like other Golgi autoantigens described earlier, is highly rich in coiled-coils . The deduced amino acid sequence included the decapeptide ESLALEELEL, which was identified as one of two signature sequences previously reported in a family of peptide hormones and neuropeptides known as "granins" . This is the first report of a Golgi complex autoantigen that bears structural similarities to the granin family of proteins. J Biol Chem, 1995 Dec 29, 270(52), 31083 - 90 A clathrin-binding site in the hinge of the beta 2 chain of mammalian AP-2 complexes; Shih W et al.; The assembly of cytosolic clathrin into the cytoplasmic face of coated pits and coated vesicles appears to be driven by the clathrin-associated protein (AP) complexes . We have previously shown that one of the large chains of the AP complexes, the beta chain, is sufficient to drive coat assembly in vitro . This chain consists of two domains, the amino-terminal trunk and the carboxyl-terminal ear, linked by a "hinge." We report here that presence of the hinge in recombinant beta trunk or in recombinant beta ear fragments is essential for driving in vitro assembly of clathrin into coats . We have also used a binding assay to map the clathrin-binding site by nested deletion of hinge sequences to a 50-residue region in the center of the hinge . This sequence is conserved in all known beta sequences from multicellular organisms . The interaction of a single beta hinge with a clathrin triskelion is weak, and we propose that recruitment of cytosolic clathrin to a forming coated pit involves simultaneous contacts between the legs of single clathrin trimers and the beta hinges of two or three membrane-bound AP complexes . Uncoating is likely to require interruption of these contacts. J Biol Chem, 1995 Dec 29, 270(52), 31071 - 6 Roles of amino acid residues surrounding phosphorylation site 1 of branched-chain alpha-ketoacid dehydrogenase (BCKDH) in catalysis and phosphorylation site recognition by BCKDH kinase; Hawes JW et al.; Branched-chain alpha-ketoacid dehydrogenase is regulated by reversible phosphorylation of serine 293 (site 1) on the E1 alpha subunit . Alanine-scanning mutagenesis was used to examine the roles of residues surrounding serine 293 in catalysis by the dehydrogenase and in substrate recognition by branched-chain alpha-ketoacid dehydrogenase kinase . Alanine substitution of serine 293 resulted in a 10-fold increased Km for alpha-ketoisovalerate, a less increased (2.8-fold) Km for alpha-ketoisocaproate, but no change in Vmax or the Km for thiamine pyrophosphate . Alanine substitutions of arginine 288, histidine 292, and aspartate 296, residues highly conserved among alpha-ketoacid dehydrogenases, resulted in inactive enzymes . Each of the inactive E1 mutants bound to the E2 core subunit with equal affinity as wild-type E1, and each produced circular dichroism spectra identical to that of wild-type E1 . Two mutations, H292A and S293E, abolished the ability of E1 apoenzyme to reconstitute with thiamine pyrophosphate . Each alanine-substituted E1 was phosphorylated at site 1 by branched-chain alpha-ketoacid dehydrogenase kinase with similar rates, with the exception of the R288A mutant, which displayed no detectable phosphorylation . Thiamine pyrophosphate inhibited the phosphorylation of all mutant enzymes with the exception of H292A, the mutant E1 that did not bind thiamine pyrophosphate. J Biol Chem, 1995 Dec 29, 270(52), 30941 - 8 Mutations in the Escherichia coli Tus protein define a domain positioned close to the DNA in the Tus-Ter complex; Skokotas A et al.; A new genetic screen for mutations in the tus gene of Escherichia coli has been devised that selects for Tus proteins with altered ability to arrest DNA replication . We report here the characterization of three such mutants: TusP42S, TusE49K, and TusH50Y . TusP42S and TusE49K arrest DNA replication in vivo at 36% of the efficiency of wild-type Tus, whereas TusH50Y functions at 78% efficiency . The loss of replication arrest activity did not correlate with changes in the stability of the Tus-TerB complexes formed by the mutant proteins . TusE49K formed a more stable protein-DNA complex than wild-type Tus (t1/2 of 178 versus 149 min, respectively) and TusP42 had a 9-min half-life, yet these two mutants showed identical efficiencies for replication arrest . When tested in vitro using a helicase assay or an oriC replication system, we observed a general, but imperfect, correlation between the in vivo and in vitro assays . Finally, the half-lives of the mutant protein-DNA complexes suggested that the domain of Tus where these mutations are located is positioned close to the DNA in the Tus-Ter complex. J Biol Chem, 1995 Dec 29, 270(52), 30927 - 32 The central aromatic residue in loop L2 of RecA interacts with DNA . Quenching of the fluorescence of a tryptophan reporter inserted in L2 upon binding to DNA; Maraboeuf F et al.; To determine the role of the central aromatic residue in one of the DNA binding domains in Escherichia coli RecA protein, we have constructed a protein in which a tryptophan fluorescence reporter is inserted in the place of phenylalanine residue 203 in loop L2, a putative DNA binding site, and measured its fluorescence . The modified protein is active both in vivo and in vitro . The binding of nucleotide cofactor (ATP or its analog adenosine 5'-O-3-thiotriphosphate) does not modify the fluorescence . By contrast, the binding of DNA, both in the absence and presence of cofactor, strongly decreases the fluorescence in intensity (40-65%) and shifts the emission peak from 344 to 337 nm . The change occurs both with single- and double-stranded DNA and also upon the binding of a second single-stranded DNA . The results indicate that the residue 203 is in fact close to the first and second DNA binding sites . However, the quenching is not total and depends only slightly on the nature of DNA bases, thus suggesting an indirect interaction with DNA bases. J Biol Chem, 1995 Dec 29, 270(52), 30862 - 8 Stepwise movement of preproteins in the process of translocation across the cytoplasmic membrane of Escherichia coli; Uchida K et al.; Derivatives of proOmpA possessing the second cysteine residue at position +302 and the first one at different positions were constructed at the DNA level . They were oxidized to form disulfide-bridged loops of different sizes at different positions . In the presence of a protonmotive force, proOmpAs possessing a smaller loop could be translocated across the membrane in vitro, whereas ones possessing loops comprising more than 16 amino acid residues were hard to translocate . The sizes of polypeptide chains that had been translocated and had become protease-resistant were determined in both the presence and absence of the protonmotive force . The size was the same for all proOmpAs possessing the first cysteine residue between +244 (proOmpA L59) and +274 (proOmpA L29) . When the first cysteine residue was moved further away from the N terminus, a sudden increase in size, of approximately 30 amino acid residues, was observed, the size being the same for proOmpAs possessing the first cysteine residue between +278 (proOmpA L25) and +293 (proOmpA L10) . The shift in size between proOmpA L29 and proOmpA L25 was observed with different proteases exhibiting different substrate specificities . Treatment with these proteases resulted in complete digestion of SecA on everted membrane vesicles, whereas Sec proteins integrated into membranes were considerably resistant to the treatment . These results can be best interpreted as that the translocation of preproteins through the secretory machinery takes place in every 30 amino acid residues and that SecA is responsible for the stepwise movement. FEBS Lett, 1995 Dec 27, 377(3), 481 - 4 Differential effects of molecular chaperones on refolding of homologous proteins; Widmann M et al.; Three homologous aspartate aminotransferases with virtually identical spatial structures and pairwise amino acid sequence identities of > 40% differ markedly with respect to the yield of renaturation upon dilution from 6 M guanidine hydrochloride (mitochondrial << cytosolic < Escherichia coli) . The enzymes also respond differently to molecular chaperones . GroEL/GroES, the Hsp60 homolog of E . coli, increased considerably the yield of renaturation of mitochondrial aspartate aminotransferase and to a lesser extent that of its cytosolic counterpart, but not that of the E . coli enzyme . DnaK/DnaJ/GrpE, the Hsp70 system of E . coli, also increased the yield of renaturation of mitochondrial aspartate aminotransferase . Apparently, specific features in the amino acid sequence or the folding pathway which are independent of the final secondary and tertiary structure determine the interactions of the folding proteins with the chaperone systems. FEBS Lett, 1995 Dec 27, 377(3), 475 - 80 A new family of lipolytic plant enzymes with members in rice, arabidopsis and maize; Brick DJ et al.; We have noted a striking similarity between the sequences of proteins in a novel family of lipases we recently reported {Upton, C . and Buckley, J . T . (1995) Trends Biol . Sci . 20, 178-9} and more than 120 sequences from the database of Expressed Sequence Tags (dbEST) which correspond to at least 30 unique genes from arabidopsis, rice and maize . A cDNA (Arab-1) corresponding to one of these sequences was isolated, sequenced and translated . There was significant similarity to sequences in the new lipase family over the entire open reading frame of Arab-1 and when expressed in E . coli, the gene product was lipolytic . Arab-1 and genes for some of the other plant proteins appear to be differentially expressed . They may play a role in the regulation of lipid metabolism during plant development. FEBS Lett, 1995 Dec 27, 377(3), 461 - 4 Induction of inducible nitric oxide synthase expression by neopterin in vascular smooth muscle cells; Schobersberger W et al.; The pteridine compounds neopterin and 7,8-dihydroneopterin serve as valuable indicators for the stimulation of the cellular immune system . Whether they exhibit distinct biochemical functions in the immunological process is at present under discussion . We show that neopterin, but not 7,8-dihydroneopterin, is a stimulus for iNOS gene expression in rat vascular smooth muscle cells in vitro . At a concentration of 20 microM, neopterin leads to an iNOS mRNA expression of 2.5 amol iNOS cDNA/micrograms total RNA . When cells were coincubated with 20 microM neopterin and 5 micrograms/ml lipopolysaccharide derived from Escherichia coli, at least an additive effect on iNOS mRNA expression could be detected (iNOS cDNA concentration was 5.0 amol/micrograms total RNA) . We speculate that neopterin enhances the macrophage-induced extracellular toxicity . This might be of relevance in situations associated with excessive release of cytokines, neopterin, and nitric oxide, as observed in septic shock. FEBS Lett, 1995 Dec 27, 377(3), 421 - 5 5'-AMP inhibits dephosphorylation, as well as promoting phosphorylation, of the AMP-activated protein kinase . Studies using bacterially expressed human protein phosphatase-2C alpha and native bovine protein phosphatase-2AC; Davies SP et al.; Human protein phosphatase-2C alpha (PP2C alpha) was purified to homogeneity after expression in Escherichia coli . AMP inhibited the dephosphorylation of AMP-activated protein kinase (AMPK), but not phosphocasein, by PP2C alpha . The concentration dependence and the effects of other nucleotides (ATP and formycin A-5'-monophosphate) suggest that AMP acts by binding to the same site which causes direct allosteric activation of AMPK . A similar, although less pronounced, effect was observed with another protein phosphatase (PP2AC) . We have now shown that AMPK activates the AMPK cascade by four mechanisms, which should make the system exquisitely sensitive to changes in AMP concentration. FEBS Lett, 1995 Dec 27, 377(3), 295 - 300 Protein phosphatase 1 interacts with p53BP2, a protein which binds to the tumour suppressor p53; Helps NR et al.; The p53 binding protein, termed p53BP2, was identified as a protein interacting with protein phosphatase 1 (PP1) in the yeast two hybrid system . The interaction was confirmed by co-immunoprecipitation of p53BP2 with epitope-tagged PP1 in vitro . The p53BP2-PP1 complex was stable to NaCl at concentrations which dissociate the p53-p53BP2 complex, and the binding of PP1 and p53 to p53BP2 was mutually exclusive . The region required for interaction with PP1 was shown to be contained within amino acids 297-431 of p53BP2, which includes two ankyrin repeats . The phosphorylase phosphatase activity of PP1 was inhibited by p53BP2 at nanomolar concentrations . These results suggest that PP1 may be involved in dephosphorylation and regulation of p53 through interaction with p53BP2. Biochim Biophys Acta, 1995 Dec 27, 1264(3), 388 - 96 Characterization of human E4BP4, a phosphorylated bZIP factor; Chen WJ et al.; In this report we described the isolation of transcription factor E4BP4 by lambda gt11 expression cloning using a probe containing the CRE/ATF-like sequence located between -2764 bp and -2753 bp in the upstream regulatory region for the human IL-1 beta gene . DNaseI protection, gel mobility shift analysis, and cotransfection studies were performed to investigate the binding and functional properties of E4BP4 using IL-1 beta promoter sequences . By DNaseI footprinting, a protection pattern was generated over the CRE/ATF-like site and the flanking sequences by bacterially produced E4BP4 . Competition experiment by gel shift assay indicated that E4BP4 bound specifically to CRE/ATF-like site, not NF kappa B-like site . In cotransfection studies, E4BP4 repressed promoter activity and this repression was mediated through the CRE/ATF-like site . Mutational analysis of E4BP4 suggested that the DNA binding as well as repression activities required leucine heptad repeat domain . Analysis of E4BP4 produced in Escherichia coli and Sf9 cells infected with recombinant baculovirus indicated that baculovirus produced protein showed enhanced binding to the CRE/ATF-like site compared to the E . coli-produced protein . Analysis of posttranslational modifications indicated that E4BP4 produced in Sf9 cells was phosphorylated and this phosphorylation was important for the DNA binding activity of E4BP4. Biochim Biophys Acta, 1995 Dec 27, 1264(3), 347 - 56 Cloning, sequence analysis and expression of mammalian mitochondrial protein synthesis elongation factor Tu; Woriax VL et al.; The bovine liver mitochondrial protein synthesis elongation factor Tu.Ts complex (EF-TU.Tsmt) has been purified and partial peptide sequence information has been obtained for EF-Tumt . A complete cDNA has been obtained encoding bovine EF-Tumt and a nearly complete cDNA has been obtained for human EF-Tumt . The bovine cDNA has a 5' untranslated leader, an open reading frame of 1356 nucleotides and a 3' untranslated region of 189 base pairs . NH2-terminal sequencing of the mature protein indicates that the transit peptide for the mitochondrial localization of this protein is 43 amino acids in length . The human and bovine factors are 95% identical . The deduced protein sequences show considerable identity to bacterial and organellar EF-Tu sequences . At least two genes for EF-Tumt are present in the bovine system . Northern analysis indicates that EF-Tumt is synthesized in all tissues but that the level of expression varies over a wide range . EF-TUmt has been expressed in E . coli as a His-tagged protein and purified to near homogeneity . The expressed form of the factor is active in the poly(U)-directed polymerization of phenylalanine although it is less active than the native EF-Tu.Tsmt complex. Biochim Biophys Acta, 1995 Dec 27, 1264(3), 330 - 6 Characterization of the binding of HU and IHF, homologous histone-like proteins of Escherichia coli, to curved and uncurved DNA; Shimizu M et al.; The binding of E . coli histone-like protein HU to curved and uncurved DNA fragments containing adenine tracts was characterized by relative binding affinity assay, and compared with that of other homologous histone-like protein integration host factor (IHF) . Both HU and IHF have about 3- to 5-fold higher affinity for overall curved DNA fragments such as (A6N4)11 and (A3T3N4)12 compared to a standard duplex fragment with mixed sequence . The binding manner of HU to the curved fragments was highly cooperative . However, loss of overall curvature for shorter fragments (< approximately 100 bp) reduced the preference of HU binding to curved (A3T3N4)n over uncurved (T3A3N4)n, indicating that the binding specificity of HU to curved DNA is length-dependent . Thus, the curved DNA configuration of the whole molecule facilitates the binding of several HU molecules to form the hierarchy of HU-DNA complex . Furthermore, it was shown that HU and IHF bind less well to (A6N9)n, which has a zig-zag straight structure, whereas they preferentially bind to uncurved (T3A3N4)14 . These results suggested that not only intrinsically overall curvature but also the preferred orientations for DNA bending in the protein-DNA complex are important factors for affinities of HU and IHF. Biochim Biophys Acta, 1995 Dec 27, 1264(3), 323 - 9 Formation of a free radical of the sulfenylimine type in the mouse ribonucleotide reductase reaction with 2'-azido-2'-deoxycytidine 5'-diphosphate; Behravan G et al.; Mouse and Escherichia coli ribonucleotide reductases (RR) both belong to the same class of RR, where the enzyme consists of two non-identical subunits, proteins R1 and R2 . A transient free radical was observed by EPR spectroscopy in the mouse RR reaction with the suicidal inhibitor 2'-azido-2'-deoxycytidine 5'-diphosphate . The detailed hyperfine structure of the EPR spectrum of the transient radical is somewhat different for the mouse and previously studied E . coli enzymes . When the positive allosteric effector ATP was replaced by the negative effector dATP, no transient radical was observed, showing that 'normal' binding of the inhibitor to the substrate binding site is required . Using the mouse protein R2 mutants W103Y and D266A, where the mutations have been shown to specifically block long range electron transfer between the active site of the R1 protein to the iron/radical site in protein R2, no evidence of transient radical was found . Taken together, the data suggest that the radical is located at the active site in protein R1, and is probably of the sulfenylimine type. Biochim Biophys Acta, 1995 Dec 27, 1264(3), 303 - 11 Expression of recombinant elongation factor 1 beta from rabbit in Escherichia coli . Phosphorylation by casein kinase II; Chen CJ et al.; The beta subunit of eukaryotic elongation factor 1 (EF-1) catalyzes the GDP/GTP exchange activity on EF-1 alpha . In these studies, two cDNAs for the beta subunit of EF-1 from rabbit are cloned and sequenced . The cDNAs consist of 808 and 798 bp and are identical except for the 5' leader sequences of 67 and 57 bp . Both cDNAs code for a protein of 225 amino acids . Using the pT7-7 expression vector, EF-1 beta was expressed in Escherichia coli and purified to apparent homogeneity by chromatography on DEAE-cellulose and FPLC on Superose 12 and Mono Q . EF-1 beta was highly phosphorylated by casein kinase II, with up to 1.3 mol of phosphate incorporated per mol protein . From microsequence analysis and manual Edman degradation, the majority of the phosphate was shown to be present in serine 106 in the peptide DLFGS106DDEEES112EEA . Serine 112 was also phosphorylated by casein kinase II, but to a lesser extent . Previously, little phosphorylation of the beta subunit by casein kinase II was observed in native EF-1 unless GDP was bound to the alpha subunit (Palen, E., Venema, R.C., Chang, Y-W.E . and Traugh, J.A . (1994) Biochemistry, 8515-8520) . In contrast, purified recombinant EF-1 beta was highly and specifically phosphorylated by casein kinase II; GDP and polylysine had little effect on the rate of phosphorylation of the purified subunit. Biochem Biophys Res Commun, 1995 Dec 26, 217(3), 940 - 9 Sequence analysis of one major basic beta-crystallin (beta Bp) of amphibian lenses: evolutionary comparison and phylogenetic relatedness between beta- and gamma-crystallins; Pan FM et al.; beta Bp-Crystallin, a major basic beta-crystallin of vertebrate eye lens, is developmentally regulated during the process of amphibian metamorphosis . In order to facilitate the determination of the primary sequence of this ubiquitous crystallin present in all vertebrate species, cDNA mixture was synthesized from the poly(A)+ mRNA isolated from bullfrog eye lenses . A protocol of Rapid Amplification of cDNA Ends (RACE) was used to amplify cDNAs encoding beta Bp-crystallin by polymerase chain reaction (PCR) . PCR-amplified product corresponding to beta Bp-crystallin was then ligated into pGEM-T vector and then transformed into E . coli strain JM109 . One complete full-length reading frame of 615 base pairs, which covers a deduced protein sequence of 205 amino acids, including the universal initiating methionine, was revealed by automatic nucleotide sequencing with a fluorescence-based dideoxynucleotide chain-termination method . It shows 83, 74, 78 and 80 percent sequence similarity to the homologous beta 2 crystallins of chicken, rat, bovine, and human species, respectively, revealing the close structural relationship among beta Bp-crystallins even from remotely related species . In this study phylogenetic trees based on nucleotide and protein sequences of various beta- and gamma-crystallins from different vertebrate classes are constructed using a combination of distance matrix and approximate parsimony methods, which corroborate the previous supposition that beta- and gamma-crystallins form a superfamily with a common ancestry. Biochem Biophys Res Commun, 1995 Dec 26, 217(3), 1223 - 30 Identification and localization of gene expression of a low M(r) GTP-binding protein, ram p25 in pituitary gland; Nagata K et al.; The localization of gene expression of a low M(r) GTP-binding protein, ram p25, was examined throughout the entire brain of adult rat by in situ hybridization . ram-mRNA was expressed predominantly in anterior and intermediate lobes of pituitary gland (hypophysis), and weakly in hippocampal formation . In other parts of brain, no significant expression of ram-mRNA was detected, indicating that ram p25 may have an important role(s) in adenohypophysis . In 15-day-fetal rat, the mRNA was observed to be expressed strongly in the primitive anterior lobe of pituitary gland . The ram p25 was also detected in bovine pituitary gland by a specific antibody and was purified to near homogeneity by stepwise column chromatographies . Biochemical characterization of the purified ram p25 revealed that the profile of {35S}GTP gamma S-binding activity was almost the same as that of recombinant ram p25 expressed in E . coli. Biochem Biophys Res Commun, 1995 Dec 26, 217(3), 1177 - 84 P-azidoiodoacetanilide, a new short photocrosslinker that has greater cysteine specificity than p-azidophenacyl bromide and p-azidobromoacetanilide; Zhang J et al.; An important criterion for a protein modifying agent is its residue selectivity . We report the synthesis of a new photocrosslinking agent, p-azidoiodoacetanilide (AIA), which has a greater specificity to modify cysteine residues than the widely used p-azidophenacyl bromide (APB) . Crosslinking of UmuD protein, which only has one cysteine, in the homodimer using APB or AIA resulted in 39% and 30% crosslinking, respectively; however, crosslinking of UmuD/C24A, a derivative with no cysteines, resulted in 16% crosslinked dimer using APB but only 2% using AIA . In addition, incorporation of {2-14C}APB into UmuD/C24A was 43% the amount of incorporation into wildtype UmuD, whereas incorporation of {2-14C}AIA into UmuD/C24A was only 13% the amount incorporated into wildtype UmuD . We also examined the cysteine specificity of p-azidobromoacetanilide (ABA) and found it to be less cysteine specific than AIA. Biochemistry, 1995 Dec 26, 34(51), 16852 - 9 Kinetic mechanism of chloramphenicol acetyltransferase: the role of ternary complex interconversion in rate determination; Ellis J et al.; Chloramphenicol acetyltransferase (CAT) catalyzes the acetyl-CoA-dependent acetylation of chloramphenicol (Cm) by a ternary complex mechanism and with a random order of addition of substrates . A closer examination of the mechanism of the reaction catalyzed by the type III CAT variant (CATIII) has included the measurement of the individual rate constants by stopped-flow fluorimetry at 5 degrees C . Under all conditions employed, product release from the binary complexes in both forward and reverse reactions was found to be too slow to account for the observed overall rate of turnover for the reaction . Additional, faster routes for product release are achieved via the formation of the nonproductive ternary complexes (CAT:3-acetyl-Cm:acetyl-CoA and CAT:CoA:Cm) . The release of 3-acetyl-Cm from the binary complex is 5-fold slower than kcat (135 s-1 at 5 degrees C), whereas the dissociation rate constants of 3-acetyl-Cm from the ternary complexes with CoA and acetyl-CoA are 120 and 200 s-1, respectively . Arrhenius plots of dissociation rate constants indicate a slow release of products over a broad temperature range . Computer simulations based on the rate constants of CATIII applied to a ternary complex mechanism, assuming random order of substrate addition and product release, yielded nonlinear initial rates of product formation unless both nonproductive ternary complexes were included in the model . Simulated steady-state kinetic analyses based on the latter assumption yielded kinetic parameters that compared favorably with those determined experimentally.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1995 Dec 26, 34(51), 16770 - 80 Characterization of the active site cysteine residues of the thioredoxin-like domains of protein disulfide isomerase; Darby NJ et al.; The dithiol/disulfide active sites of each of the two isolated thioredoxin-like domains of protein disulfide isomerase (PDI) expressed in Escherichia coli have been characterized in order to understand their catalytic mechanisms and their functions in PDI . In each of the folded domains, as in other proteins of the thioredoxin family, only one of the cysteine residues of the active site sequence -Cys-Gly-His-Cys- is accessible, and its thiol group is highly reactive and has a low pKa value . The kinetics and equilibria have been measured of the reactions between the active site cysteine residues and glutathione, the predominant thiol/disulfide reagent of the endoplasmic reticulum . A disulfide bond can be formed very rapidly between the pair of cysteine residues of each domain, but each disulfide bond is very unstable and reacts rapidly with reduced glutathione . The very low stabilities of these disulfide bonds, which destabilize the protein structures, account for the efficiency with which PDI and each of the isolated domains can introduce disulfide bonds into proteins . These kinetics and equilibrium data go far in helping to understand the catalytic mechanism of PDI and its individual domains. Biochemistry, 1995 Dec 26, 34(51), 16708 - 14 Magnesium acetate induces a conformational change in Escherichia coli primase; Urlacher TM et al.; Primase from Escherichia coli is a single-stranded DNA-dependent RNA polymerase . As such, it requires magnesium to carry out catalysis . Limited tryptic digestion was used to probe the conformations of primase as a function of magnesium acetate concentration . In the absence of magnesium, trypsin cleaved primase at three sites . Magnesium acetate induced a conformational change such that one of these sites became inaccessible to trypsin digestion and a new site became trypsin accessible . The conformational change was only induced by Mg(OAc)2 and not MnCl2, CaCl2, NaOAc or LiCl, indicating a clear magnesium acetate-dependent conformational change . The effect was slightly induced by MgSO4 and MgCl2 . An allosteric binding model indicates that primase binds at least two magnesiums in a cooperative manner . The data were best fit to a two-state model in which one conformation had a high affinity for magnesium, KR = 83.4 M-1, and the other state had virtually no affinity. Biochemistry, 1995 Dec 26, 34(51), 16695 - 702 Effect of metal-ligand mutations on phosphoryl transfer reactions catalyzed by Escherichia coli glutamine synthetase; Abell LM et al.; Glutamine synthetase (GS) converts glutamate to glutamine in the presence of ATP and ammonia and requires two divalent metal ions, designated n1 and n2, for catalysis . The first intermediate, gamma-glutamyl phosphate, is formed during catalysis by the transfer of the gamma-phosphate of ATP to the gamma-carboxylate of glutamate . Efficient phosphoryl transfer between these two negatively charged moieties is thought to be mediated by the n2 metal . To explore the role of the n2 metal in catalysis, histidine 269, a ligand to the n2 metal, was changed to aspartate, asparagine, glutamate, and glutamine by site-directed mutagenesis . All of the mutants bind two manganese ions as determined by EPR titration . The mutations had little effect on the substrate Km's except in the case of H269E which exhibited a Km Glu = 92 mM, a 1000-fold increase compared to that for WT (Km Glu = 70 microM) . The ability of these mutants to catalyze phosphoryl transfer to glutamate or to the inhibitor phosphinothricin was examined by rapid quench kinetic experiments . Phosphorylated phosphinothricin was detected by 31P NMR and shown to be produced by both mutants and WT . The rate of phosphoryl transfer to PPT for H269E is reduced 100-fold (0.030 s-1) compared to WT (8 s-1) . The extra negative charge around the n2 metal ion contributed by glutamate 269 severely reduces the ability of the n2 metal to mediate efficient glutamate binding in the presence of negatively charged ATP and weakens the interactions between metal ion and the reactants in the transition state, thus resulting in a lower rate of phosphoryl transfer. Biochemistry, 1995 Dec 26, 34(51), 16624 - 31 Evidence for distinct ligand-bound conformational states of the multifunctional Escherichia coli repressor of biotin biosynthesis; Xu Y et al.; The Escherichia coli repressor of biotin biosynthesis (BirA) is a unique transcriptional repressor which catalyzes synthesis of its own corepressor and catalyzes attachment of a cofactor to an essential metabolic enzyme . BirA both catalyzes synthesis of biotinyl-5'-AMP from the substrates ATP and biotin and transfer of the biotin moiety from the adenylate to a lysine residue of a subunit of the acetyl-CoA carboxylase . BirA-bio-5'-AMP, moreover, binds sequence specifically to the biotin operator to repress transcription of the biotin biosynthetic genes . Using a combination of kinetic measurements of binding of the two ligands, biotin and bio-5'-AMP, to BirA as well as proteolytic digestion experiments, we have found evidence for at least three discrete conformational states of BirA . Results of stopped-flow fluorescence measurements of association of both ligands with BirA indicate that the process involves initial formation of a collision complex followed by a slow conformational change . The kinetics of the conformational change are distinct for the two ligands and are the basis for the difference in the thermodynamic stabilities of the two protein-ligand complexes . Different rates of proteolytic digestion of apoBirA and complexes of BirA with the two ligands were also observed . Results of the combined approaches indicate that apoBirA, and the BirA-bio-5'-AMP and BirA-biotin complexes are conformationally distinct. Biochemistry, 1995 Dec 26, 34(51), 16596 - 607 Structure of the A-domain of HMG1 and its interaction with DNA as studied by heteronuclear three- and four-dimensional NMR spectroscopy; Hardman CH et al.; HMG1 has two homologous, folded DNA-binding domains ("HMG boxes"), A and B, linked by a short basic region to an acidic C-terminal domain . Like the whole protein, which may perform an architectural role in chromatin, the individual boxes bind to DNA without sequence specificity, have a preference for distorted or prebent DNA, and are able to bend DNA and constrain negative superhelical turns . They show qualitatively similar properties with quantitative differences . We have previously determined the structure of the HMG box from the central B-domain (77 residues) by two-dimensional NMR spectroscopy, which showed that it contains a novel fold {Weir et al . (1993) EMBO J . 12, 1311-1319} . We have now determined the structure of the A-domain (as a Cys-->Ser mutant at position 22 to avoid oxidation, without effect on its DNA-binding properties or structure) using heteronuclear three- and four-dimensional NMR spectroscopy . The A-domain has a very similar global fold to the B-domain and the Drosophila protein HMG-D {Jones et al . (1994) Structure 2, 609-627} . There are small differences between A and B, in particular in the orientation of helix I, where the B-domain is more similar to HMG-D than it is to the A-domain; these differences may turn out to be related to the subtle differences in functional properties between the two domains {Teo et al . (1995) Eur . J . Biochem . 230, 943-950} and will be the subject of further investigation . NMR studies of the interaction of the A-domain of HMG1 with a short double-stranded oligonucleotide support the notion that the protein binds via the concave face of the L-shaped structure; extensive contacts with the DNA are made by the N-terminal extended strand, the N-terminus of helix I, and the C-terminus of helix II . These contacts are very similar to those seen in the LEF-1 and SRY-DNA complexes {Love et al . (1995) Nature 376, 791-795; Werner et al . (1995) Cell 81, 705-714}. Biochemistry, 1995 Dec 26, 34(51), 16585 - 95 2 A resolution structure of DppA, a periplasmic dipeptide transport/chemosensory receptor; Nickitenko AV et al.; The family of about 50 periplasmic binding proteins, which exhibit diverse specificity (e.g., carbohydrates, amino acids, dipeptides, oligopeptides, oxyanions, metals, and vitamins) and range in size from 20 to 58 kDa, is a gold mine for an atomic-level investigation of structure and molecular recognition . These proteins serve as initial receptors for active transport systems or permeases . About six of these proteins, including the dipeptide-binding protein (DppA), are also primary receptors for chemotaxis . The structure of the unbound form of DppA (M(r) = 57,400) has been determined and refined to an R-factor of 0.169 to 2 A resolution . DppA consists of two distinct domains (I and II) connected by two "hinge" segments which form part of the base of the wide groove between the two domains . The relative orientation of the two domains gives the protein a pearlike shape, with domain I and domain II forming the larger and smaller apical ends, respectively . From the tip to the rounded bottom measures about 85 A, and the widest diameter is about 60 A . Domain I, which consists of two integrated subdomains, is folded from two separate polypeptide segments from the amino- and carboxyl-terminal ends . The more compact domain II is formed from the intervening segment . Comparison of the dipeptide-binding protein structure with that of the bound form of the similar oligopeptide-binding protein {Tame, J . R . H., Murshudov, G . N., Dodson, E . J., Neil, T . K., Dodson, G . G., Higgins, C . F., & Wilkinson, A . J . (1994) Science 264, 1578-1581} reveals the major features that differentiate the ligand specificity of the two proteins and describe the large hinge bending (about 55 degrees) between the two domains. Biochemistry, 1995 Dec 26, 34(51), 16552 - 62 Folding pathway of Escherichia coli ribonuclease HI: a circular dichroism, fluorescence, and NMR study; Yamasaki K et al.; The unfolding and refolding processes of Escherichia coli ribonuclease HI at 25 degrees C, induced by concentration jumps of either guanidine hydrochloride (GuHCl) or urea, were investigated using stopped-flow circular dichroism (CD), stopped-flow fluorescence, and NMR spectroscopies . Only a single exponential process was detected for the fast time scale unfolding (rate constants from 0.014 to 0.54 s-1, depending on the final denaturant concentration) . For refolding, the far-UV CD value largely recovered within 50 ms of the stopped-flow mixing dead time (burst phase) . This phase was followed by either one or two phases, with rate constants from 0.035 to 2.45 s-1 as detected by CD and fluorescence, respectively . Although this protein has a single cis-Pro residue, a very slow phase due to proline isomerization was not observed, for either unfolding or refolding . The difference in the amplitudes of the burst phases for refolding in the far- and near-UV CD spectra revealed that an intermediate state exists, with the characteristics of a molten globule . Because the one-phased fast exponential process detected by CD corresponds to the slower of the two phases detected by fluorescence, the intermediate detected by CD might be the most stable . GuHCl denaturation experiments revealed that this intermediate cooperatively unfolds, with a transition midpoint of 1.33 +/- 0.03 M . The Gibbs free energy difference (delta G) between the intermediate and the unfolded states, under physiological conditions (25 degrees C, pH 5.5, and 0 M GuHCl), was estimated to be 20.0 +/- 2.3 kJ mol-1 . Therefore, it is reasonable to assume that the refolding intermediate, rather than the unfolded state, is the latent denatured state under physiological conditions . Approximately linear relationships between the GuHCl concentration and the logarithm of the microscopic rate constants determined by CD and fluorescence were also observed . By extrapolation to a GuHCl concentration of 0 M, activation Gibbs free energies of 98.5 +/- 1.1 kJ mol-1 for unfolding and 69.5 +/- 0.2 kJ mol-1 for refolding under physiological conditions were obtained . The hydrogen-exchange-refolding competition combined with two-dimensional NMR revealed that the amide protons of alpha-helix I are the most highly protected, suggesting that alpha-helix I is the initial site of protein folding . The CD and NMR data showed that the intermediate state has a structure similar to that of the acid-denatured molten globule. Nucleic Acids Res, 1995 Dec 25, 23(24), 5006 - 11 RNA:DNA complex formation upon transcription of immunoglobulin switch regions: implications for the mechanism and regulation of class switch recombination; Daniels GA et al.; Central the regulation and mechanism of class switch recombination is the understanding of the relationship between transcription and DNA recombination . We demonstrated previously, using mini-chromosome substrates, that physiologically oriented transcription is required for recombination to occur between switch regions . In this report, we demonstrate the formation of an RNA:DNA complex under in vitro transcription conditions for these same and other switch DNA fragments . We find that cell-free transcription of repetitive murine switch regions (Smu, S gamma 2b and S gamma 3) leads to altered DNA mobility on agarose gels . These altered mobilities are resistant to RNase A but sensitive to RNase H . Transcription in the presence of labeled ribonucleotides demonstrates the stable physical association of the RNA with the DNA . Importantly, complex formation only occurs upon transcription in the physiologic orientation . Reaban and Griffin {1990 Nature, 348, 342-344} found an RNA:DNA hybrid structure that was limited to an atypical 143 nucleotide purine region within a 2.3 kb S alpha segment . Here we demonstrate RNA:DNA hybrid formation in more typical switch sequences (lacking the atypical 143 nucleotide purine tract) from a variety of switch regions that are only 60-70% purine on the non-template strand . These results suggest a general model involving an RNA:DNA complex as an intermediate during class switch recombination. J Biol Chem, 1995 Dec 22, 270(51), 30804 - 12 Dissociation of hexameric Escherichia coli inorganic pyrophosphatase into trimers on His-136-->Gln or His-140-->Gln substitution and its effect on enzyme catalytic properties; Baykov AA et al.; Each of the five histidines in Escherichia coli inorganic pyrophosphatase (PPase) was replaced in turn by glutamine . Significant changes in protein structure and activity were observed in the H136Q and H140Q variants only . In contrast to wild-type PPase, which is hexameric, these variants can be dissociated into trimers by dilution, as shown by analytical ultracentrifugation and cross-linking . Mg2+ and substrate stabilize the hexameric forms of both variants . The hexameric H136Q- and H140Q-PPases have the same binding affinities for magnesium ion as wild-type, but their hydrolytic activities under optimal conditions are, respectively, 225 and 110% of wild-type PPase, and their synthetic activities, 340 and 140% . The increased activity of hexameric H136Q-PPase results from an increase in the rate constants governing most of the catalytic steps in both directions . Dissociation of the hexameric H136Q and H140Q variants into trimers does not affect the catalytic constants for PPi hydrolysis between pH 6 and 9 but drastically decreases their affinities for Mg2PPi and Mg2+ . These results prove that His-136 and His-140 are key residues in the dimer interface and show that hexamer formation improves the substrate binding characteristics of the active site. J Biol Chem, 1995 Dec 22, 270(51), 30773 - 80 Phosphorylation of both serine residues in cardiac troponin I is required to decrease the Ca2+ affinity of cardiac troponin C; Zhang R et al.; The phosphorylation of cardiac muscle troponin I (CTnI) at two adjacent N-terminal serine residues by cAMP-dependent protein kinase (PKA) has been implicated in the inotropic response of the heart to beta-agonists . Phosphorylation of these residues has been shown to reduce the Ca2+ affinity of the single Ca(2+)-specific regulatory site of cardiac troponin C (CTnC) and to increase the rate of Ca2+ dissociation from this site (Robertson, S . P., Johnson, J . D., Holroyde, M . J., Kranias, E . G., Potter, J . D., and Solaro, R . J . (1982) J . Biol . Chem . 257, 260-263) . Recent studies (Zhang, R., Zhao, J., and Potter, J . D . (1995) Circ . Res . 76, 1028-1035) have correlated this increase in Ca2+ dissociation with a reduced Ca2+ sensitivity of force development and a faster rate of cardiac muscle relaxation in a PKA phosphorylated skinned cardiac muscle preparation . To further determine the role of the two PKA phosphorylation sites in mouse CTnI (serine 22 and 23), serine 22 or 23, or both were mutated to alanine . The wild type and the mutated CTnIs were expressed in Escherichia coli and purified . Using these mutants, it was found that serine 23 was phosphorylated more rapidly than serine 22 and that both serines are required to be phosphorylated in order to observe the characteristic reduction in the Ca2+ sensitivity of force development seen in a skinned cardiac muscle preparation . The latter result confirms that PKA phosphorylation of CTnI, and not other proteins, is responsible for this change in Ca2+ sensitivity . The results also suggest that one of the serines (23) may be constitutively phosphorylated and that serine 22 may be functionally more important. J Biol Chem, 1995 Dec 22, 270(51), 30657 - 63 ARP is a plasma membrane-associated Ras-related GTPase with remote similarity to the family of ADP-ribosylation factors; Schurmann A et al.; The human and rat homologues of a novel Ras-related GTPase with unique structural features were cloned by polymerase chain reaction amplification and cDNA library screening . Their deduced amino acid sequences are highly homologous (97% identical amino acids; 88.3% identical nucleotides within the coding region) and comprise all six of the conserved motifs presumably involved in GTP binding . Because the sequences exhibit some similarity with members of the ADP-ribosylation factor (ARF) family (33% identity with ADP-ribosylation factor 1 (ARF1), 39% identity with ARF-like 3), the protein was designated ARP (ARF-related protein) . In contrast to all other members of the ARF family, ARP lacks the myristoylation site at position 2 and comprises an insertion of 8 amino acids in the region between PM1 and PM2 . mRNA was found in most rat tissues examined (skeletal muscle, fat, liver, kidney, spleen, testis, adrenals, ovary, thymus, intestine, and lung) . Western blot analysis with antiserum against recombinant ARP showed a 25-kDa protein in membranes from rat liver, testis, and kidney . Thus, the protein appears to be post-translationally modified for membrane anchoring . Unlike ARF, the ARP immunoreactivity was detected in plasma membranes but not in cytosol of fractionated 3T3-L1 cells . Recombinant ARP exhibited specific and saturable GTP gamma S (guanosine 5'-3-O-(thio)triphosphate) binding and, unlike ARF isotypes, GTPase activity in the absence of tissue extracts or phospholipids . Thus, the structural and functional characteristics of ARP indicate that it represents a novel subtype of GTPases, presumably exerting a unique function and possibly involved in plasma membrane-related signaling events. J Biol Chem, 1995 Dec 22, 270(51), 30545 - 50 Site-directed mutagenic alteration of potential active-site residues of the A subunit of Escherichia coli heat-labile enterotoxin . Evidence for a catalytic role for glutamic acid 112; Cieplak W Jr et al.; Escherichia coli heat-labile enterotoxin (LT) and the related cholera toxin exert their effects on eukaryotic cells through the ADP-ribosylation of guanine nucleotide-binding proteins of the adenylate cyclase complex . The availability of the crystal structure for LT has permitted the tentative identification of residues that lie within or are vicinal to a presumptive NAD(+)-binding site and thus may play a role in substrate binding or catalysis . Using a plasmid clone encoding the A subunit of LT, we have introduced substitutions at such potential active-site residues and analyzed the enzymatic properties of the resultant mutant analogs . Enzymatic analyses, employing both transducin and agmatine as acceptor substrates, revealed that substitutions at serine 61, glutamic acid 110, and glutamic acid 112 resulted in reduction of enzyme activity to < 10% of wild-type levels . Kinetic analyses indicated that alteration of these sites affected the catalytic rate of the enzyme and had little or no effect on the binding of either NAD+ or agmatine . Of the mutant analogs analyzed, only glutamic acid 112 appeared to represent an essential catalytic residue as judged by the relative effects on kcat and kcat/Km . The results provide formal evidence that glutamic acid 112 of the A subunit of LT represents a functional homolog or analog of catalytic glutamic acid residues that have been identified in several other bacterial ADP-ribosylating toxins and that it may play an essential role in rendering NAD+ susceptible to nucleophilic attack by an incoming acceptor substrate. J Biol Chem, 1995 Dec 22, 270(51), 30532 - 44 Spectroscopic and kinetic properties of unphosphorylated rat hepatic phenylalanine hydroxylase expressed in Escherichia coli . Comparison of resting and activated states; Kappock TJ et al.; The non-heme iron-dependent metalloenzyme, rat hepatic phenylalanine hydroxylase (EC 1.14.16.1; phenylalanine 4-monooxygenase (PAH) was overexpressed in Escherichia coli and purified to homogeneity, allowing a detailed comparison of the kinetic, hydrodynamic, and spectroscopic properties of its allosteric states . The homotetrameric recombinant enzyme, which is highly active and contains 0.7-0.8 iron atoms per subunit, is identical to the native enzyme in several properties: Km, 6-methyltetrahydropterin = 61 microM and L-Phe = 170 microM; Vmax = 9 s-1 (compared to 45 microM, 180 microM, and 13 s-1 for the rat hepatic enzyme) . L-Phe and lysolecithin treatment induce the rPAHT-->rPAHR (where r is recombinant) allosteric transformation necessary for rPAH activity . Characteristic changes in the fluorescence spectra, increased hydrophobicity, a large activation energy barrier, and a 10% volume increase of the tetrameric structure are consistent with a significant reorganization of the protein following allosteric activation . However, optical and EPR spectroscopic data suggest that only minor changes occur in the primary coordination sphere (carboxylate/histidine/water) of the catalytic iron center . Detailed steady state kinetic investigations, using 6-methyltetrahydropterin as cofactor and lysolecithin as activator, indicate rPAH follows a sequential mechanism . A catalytic Arrhenius Eact of 14.6 +/- 0.3 kcal/mol subunit was determined from temperature-dependent stopped-flow kinetics data . rPAH inactivates during L-Phe hydroxylation with a half-life of 4.3 min at 25 degrees C, corresponding to an Arrhenius Eact of 10 +/- 1 kcal/mol subunit for the inactivation process . Catechol binding (2.4 x 10(6) M-1) is shown to occur only at catalytically competent iron sites . Ferrous rPAH binds NO, giving rise to an ST = 3/2 spin system. J Biol Chem, 1995 Dec 22, 270(51), 30508 - 15 The C-terminal region of the UvrB protein of Escherichia coli contains an important determinant for UvrC binding to the preincision complex but not the catalytic site for 3'-incision; Moolenaar GF et al.; The UvrABC endonuclease from Escherichia coli repairs damage in the DNA by dual incision of the damaged strand on both sides of the lesion . The incisions are in an ordered fashion, first on the 3'-side and next on the 5'-side of the damage, and they are the result of binding of UvrC to the UvrB-DNA preincision complex . In this paper, we show that at least the C-terminal 24 amino acids of UvrB are involved in interaction with UvrC and that this binding is important for the 3'-incision . The C-terminal region of UvrB, which shows homology with a domain of the UvrC protein, is part of a region that is predicted to be able to form a coiled-coil . We therefore propose that UvrB and UvrC interact through the formation of such a structure . The C-terminal region of UvrB only interacts with UvrC when present in the preincision complex, indicating that the conformational change in UvrB accompanying the formation of this complex exposes the UvrC binding domain . Binding of UvrC to the C-terminal region of UvrB is not important for the 5'-incision, suggesting that for this incision a different interaction of UvrC with the UvrB-DNA complex is required . Truncated UvrB mutants that lack up to 99 amino acids from the C terminus still give rise to significant incision (1-2%), indicating that this C-terminal region of UvrB does not participate in the formation of the active site for 3'-incision . This region, however, contains the residue (Glu-640) that was proposed to be involved in 3'-catalysis, since a mutation of the residue (E640A) fails to promote 3'-incision (Lin, J.J., Phillips, A.M., Hearst, J.E., and Sancar, A . (1992) J . Biol . Chem . 267, 17693-17700) . We have isolated a mutant UvrB with the same E640A substitution, but this protein shows normal UvrC binding and incision in vitro and also results in normal survival after UV irradiation in vivo . As a consequence of these results, it is still an open question as to whether the catalytic site for 3'-incision is located in UvrB, in UvrC, or is formed by both proteins. J Biol Chem, 1995 Dec 22, 270(51), 30499 - 507 The Y-box motif mediates redox-dependent transcriptional activation in mouse cells; Duh JL et al.; We show here that the OxyR response element (ORE) in the bacterial oxyR promoter can also function as a redox-dependent enhancer in mammalian cells . Fusion of ORE to an SV40 basal promoter driving chloramphenicol acetyltransferase (CAT) expression confers H2O2 inducibility to expression of the cat gene in mouse Hepa-1 hepatoma cells . Nuclear extracts from these cells contain DNA-binding proteins that specifically interact with ORE DNA, cannot be completed by cognate oligonucleotides to AP-1 or NF kappa B, and are constitutively expressed, since treatment with H2O2 causes no detectable changes in binding activity or DNA-protein interaction . Recombinant cDNA clones that express ORE-binding proteins were isolated from a mouse hepatoma expression library and found to be representatives of two different members of the murine Y-box family of transcription factors . Canonical Y-box and ORE oligonucleotides compete with each other for binding to Y-box proteins in gel shift assays and antibodies to FRGY2, a Xenopus Y-box protein, supershift both Y-box and ORE DNA-protein complexes . In addition, antisense oligonucleotides to mouse YB-1 mRNA abolish induction of ORE-mediated cat expression by H2O2, and luciferase reporter constructs containing ORE, or the Y-box from the human MHC class II HLA-DQ gene, exhibit identical dose-dependent H2O2 inducibilities, which can be abolished by addition of 2-mercaptoethanol to the culture medium . These results suggest that the Y-box proteins may be an integral component of a eukaryotic redox signaling pathway. J Biol Chem, 1995 Dec 22, 270(51), 30491 - 8 Processive post-translational modification . Vitamin K-dependent carboxylation of a peptide substrate; Morris DP et al.; Mass spectrometry has been used to demonstrate that vitamin K-dependent carboxylation is a processive post-translational modification (i.e . multiple carboxylations occur during a single association between enzyme and substrate) . Purified vitamin K-dependent carboxylase can carboxylate as many as 12 glutamate residues in FIXQ/S, a peptide substrate based on amino acids -18 to 41 of the human blood clotting enzyme factor IX . Mass spectrometry was used to determine the number of gamma-carboxyl groups added to FIXQ/S by the carboxylase during an in vitro reaction . Despite the fact that most substrate molecules in a reaction were uncarboxylated, almost all carboxylated FIXQ/S molecules were carboxylated many times . This observation can only be explained by two types of mechanisms . In a processive mechanism, multiple carboxylations could occur during a single substrate binding event . Alternatively, a distributive mechanism could result in the observed behavior if the initial carboxylation event results in a substrate that is additionally carboxylated far more efficiently than the uncarboxylated FIXQ/S . Kinetic experiments and arguments were used to show that the vitamin K-dependent carboxylase is not distributive but rather is one of the first well documented examples of an enzyme that catalyzes a processive post-translation modification. J Biol Chem, 1995 Dec 22, 270(51), 30470 - 8 Purification, characterization, and molecular cloning of a novel rat liver Dopa/tyrosine sulfotransferase; Sakakibara Y et al.; A novel sulfotransferase was purified from the rat liver cytosol to electrophoretic homogeneity via five column chromatography steps (hydroxylapatite I, DEAE Bio-Gel, ATP-agarose I, hydroxylapatite II, and ATP-agarose II) . The minimum molecular weight of the purified enzyme was determined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis to be approximately 33,000 . Gel filtration chromatography revealed a native molecular weight of approximately 34,000, indicating the enzyme being present in the monomeric form . The purified sulfotransferase displayed enzymatic activities, with a pH optimum of 9.25, toward various tyrosine and 3,4-dihydroxyphenylalanine (Dopa) isomers, except DL-ortho-tyrosine . Thyroid hormones, as well as dopamine and p-nitrophenol, could also be used as substrates . The apparent Km value of the enzyme (designated the Dopa/tyrosine sulfotransferase) for L-Dopa, determined at a constant 14 microM of 3'-phosphoadenosine 5'-phosphosulfate, was 0.76 mM . The intact enzyme was found to be N-blocked when subjected to N-terminal sequencing . Three internal partial amino acid sequences, obtained by analyzing its proteolytic fragments, were found to be distinct from the homologous sequences of other known rat liver sulfotransferases . The deduced amino acid sequence of a full-length cDNA isolated from a rat liver cDNA library confirmed the identity of the Dopa/tyrosine sulfotransferase as a new type of aryl sulfotransferase . Upon transfection of COS-7 cells with an expression vector (pMSG-CMV) harboring the full-length cDNA, a 33-kDa protein displaying enzymatic and immunological properties similar to those of the purified Dopa/tyrosine sulfotransferase was expressed. J Biol Chem, 1995 Dec 22, 270(51), 30458 - 63 Identification of amino acid residues critical for catalysis and cosubstrate binding in the flavonol 3-sulfotransferase; Marsolais F et al.; The comparison of the deduced amino acid sequences of plant and animal sulfotransferases (ST) has allowed the identification of four well conserved regions, and previous experimental evidence suggested that regions I and IV might be involved in the binding of the cosubstrate, 3'-phosphoadenosine 5'-phosphosulfate (PAPS) . Moreover, region IV is homologous to the glycine-rich phosphate binding loop (P-loop) motif known to be involved in nucleotide phosphate binding in several protein families . In this study, the function of amino acid residues within these two regions was investigated by site-directed mutagenesis of the plant flavonol 3-ST . In region I, our results identify Lys59 as critical for catalysis, since replacement of this residue with alanine resulted in a 300-fold decrease in specific activity, while a 15-fold reduction was observed after the conservative replacement with arginine . Photoaffinity labeling of K59R and K59A with {35S}PAPS revealed that Lys59 is not required for cosubstrate binding . However, the K59A mutant had a reduced affinity for 3'-phosphoadenosine 5'-phosphate (PAP)-agarose, suggesting that Lys59 may participate in the stabilization of an intermediate during the reaction . In region IV, all substitutions of Arg276 resulted in a marked decrease in specific activity . Conservative and unconservative replacements of Arg276 resulted in weak photoaffinity labeling with {35S}PAPS and the R276A/T73A and R276E enzymes displayed reduced affinities for PAP-agarose, suggesting that the Arg276 side chain is required to bind the cosubstrate . The analysis of the kinetic constants of mutant enzymes at residues Lys277, Gly281, and Lys284 allowed to confirm that region IV is involved in cosubstrate binding. J Biol Chem, 1995 Dec 22, 270(51), 30401 - 7 A mutation in the ATP binding domain of rho alters its RNA binding properties and uncouples ATP hydrolysis from helicase activity; Pereira S et al.; The Escherichia coli mutant rho201 was originally isolated in a genetic screen for defects in rho-dependent termination . Cloning and sequencing of this gene reveals a single phenylalanine to cysteine mutation at residue 232 in the ATP binding domain of the protein . This mutation significantly alters its RNA binding properties so that it binds trp t', RNA 100-fold weaker than the wild type protein, with a Kd of approximately 1.3 nM . Rho201 binds nonspecific RNA only 3-4-fold less tightly than it binds trp t', while the wild type differential for these same RNAs is 10-20-fold . Curiously, rho201 displays increased secondary site RNA activation, with a Km for ribo(C)10 of 0.6 microM, compared to the wild type value of 3-4 microM . Although rho201 and the wild type protein hydrolyze ATP similarly with poly(C), or trp t' RNA, as cofactors, rho201 has a higher ATPase activity when activated by nonspecific RNA . Physically, rho201 displays an abnormal conformation detectable by mild trypsin digestion . Despite effective ATP hydrolysis, the rho201 mutant is a poor RNA:DNA helicase and terminates inefficiently on trp t' . The single F232C mutation thus appears to uncouple the protein's ATPase activity from its helicase function, so rho can no longer harness available energy for use in subsequent reactions. J Biol Chem, 1995 Dec 22, 270(51), 30392 - 400 The mechanism and substrate specificity of the NADPH:flavin oxidoreductase from Escherichia coli; Fieschi F et al.; The NAD(P)H:flavin oxidoreductase from Escherichia coli, Fre, is a monomer of 26.2 kDa that catalyzes the reduction of free flavins by NADPh or NADH . Overexpression in E . coli now allows the preparation of large amounts of pure protein . Structural requirements for recognition of flavins as substrates and not as cofactors were studied by steady-state kinetics with a variety of flavin analogs . The entire isoalloxazine ring was found to be the essential part of the flavin molecule for interaction with the polypeptide chain . Methyl groups at C-7 and C-8 of the isoalloxazine ring and the N-3 of riboflavin also play an important role in that interaction, whereas the ribityl chain of the riboflavin is not required for binding to the protein . On the other hand, the presence of the 2'-OH of the ribityl chain stimulates the NADPH-dependent reaction significantly . Moreover, a study of competitive inhibitors for both substrates demonstrated that Fre follows a sequential ordered mechanism in which NADPH binds first followed by riboflavin . Lumichrome, a very good inhibitor of Fre, may be used to inhibit flavin reductase in E . coli growing cells . As a consequence, it can enhance the antiproliferative effect of hydroxyurea, a cell-specific ribonucleotide reductase inactivator. J Biol Chem, 1995 Dec 22, 270(51), 30384 - 91 The envA permeability/cell division gene of Escherichia coli encodes the second enzyme of lipid A biosynthesis . UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase; Young K et al.; The envA gene of Escherichia coli has been shown previously to be essential for cell viability (Beall, B . and Lutkenhaus, J . (1987) J . Bacteriol . 169, 5408-5415), yet it encodes a protein of unknown function . Extracts of strains harboring the mutant envA1 allele display 3.5-18-fold reductions in UDP-3-O-acyl-N-acetylglucosamine deacetylase specific activity . The deacetylase is the second enzymatic step of lipid A biosynthesis . The structural gene coding for the deacetylase has not been assigned . In order to determine if the envA gene encodes the deacetylase, envA was cloned into an isopropyl-1-thio-beta-D-galactopyranoside-inducible T7-based expression system . Upon induction, a protein of the size of envA was highly overproduced, as judged by SDS-PAGE . Direct deacetylase assays of cell lysates revealed a concomitant approximately 5,000-fold overproduction of activity . Assays of the purified, overproduced EnvA protein demonstrated a further approximately 5-fold increase in specific activity . N-terminal amino acid sequencing of the purified protein showed that the first 20 amino acids matched the predicted envA nucleotide sequence . Contaminating species were present at less than 1% of the level of the EnvA protein . Thus, envA is the structural gene for UDP-3-O-acyl-GlcNAc deacetylase . Based on its function in lipid A biosynthesis, we propose the new designation lpxC for this gene. J Biol Chem, 1995 Dec 22, 270(51), 30274 - 7 Cysteine-rich region of Raf-1 interacts with activator domain of post-translationally modified Ha-Ras; Hu CD et al.; The interaction between "switch I/effector domain" of Ha-Ras and the Ras-binding domain (RBD, amino acid 51-131) of Raf-1 is essential for signal transduction . However, the importance of the "activator domain" (approximately corresponding to amino acids 26-28 and 40-49) of Ha-Ras and of the "cysteine-rich region" (CRR, amino acids 152-184) of Raf-1 have also been proposed . Here, we found that Raf-1 CRR interacts directly with Ha-Ras independently of RBD and that participation of CRR is necessary for efficient Ras-Raf binding . Furthermore, Ha-Ras carrying mutations (N26G and V45E) in the activator domain failed to bind CRR, whereas they bound RBD normally . On the contrary, Ha-Ras carrying mutations in the switch I/effector domain exhibited severely reduced ability to bind RBD, whereas their ability to bind CRR was unaffected . Mutants that bound to either RBD or CRR alone failed to activate Raf-1 . Ha-Ras without post-translational modifications, which lacks the ability to activate Raf-1, selectively lost the ability to bind CRR . These results suggest that the activator domain of Ha-Ras participates in activation of Raf-1 through interaction with CRR and that post-translational modifications of Ha-Ras are required for this interaction. J Biol Chem, 1995 Dec 22, 270(51), 30268 - 70 The C-terminal sequence of the chaperonin GroES is required for oligomerization; Seale JW et al.; The Escherichia coli protein GroES is a co-chaperonin that is able to assist GroEL in the refolding of proteins . GroES is a heptamer of seven identical subunits . Recent work has focused on the structural aspects of GroES . We have investigated the role of the C-terminal portion of GroES on its oligomerization . Limited proteolysis of GroES by carboxypeptidase Y gives a product in which the C-terminal 7 amino acid residues have been removed . Sedimentation velocity analysis reveals that the truncated form of GroES is unable to reassemble . The results presented here implicate the C-terminal sequence in intermonomer actions within the GroES oligomer . In addition, this work provides experimental verification of predictions implied in the recent x-ray determination of the GroES structure (Hunt, J . F., Weaver, A . J., Landry, S . J., Gierasch, L . M., and Deisenhofer, J . Nature, in press). Arch Biochem Biophys, 1995 Dec 20, 324(2), 391 - 400 Epitope mapping and tight-binding inhibition with monoclonal antibodies directed against Escherichia coli glucosamine 6-phosphate synthase; Cochet O et al.; In the present work, we attempt to identify inhibitory monoclonal antibodies directed against Escherichia coli glucosamine-6P synthase (GlmS) and to localize the corresponding epitopes to better understand the topology of the enzyme during catalysis . Four of the 15 monoclonal antibodies have been shown to be specific for the native form of the enzyme and 2 of them, 505.1 and 522.2, strongly inhibit the glucosamine synthase activity . Kinetic analysis of 505.1 antibody behavior revealed a tight-binding inhibition with a Ki = 40 +/- 20 pM, a value which is four orders of magnitude lower than the best active site-directed inhibitor reported so far . The reactivity of all the monoclonal antibodies with 601 overlapping octapeptides covering the entire sequence of GlmS was tested by enzyme-linked immunosorbent assay for precise epitope mapping . Four linear epitopes specific for the denatured protein and one present in both native and denatured enzyme were defined by this approach . Neither 505.1 nor 522.2 was directed against linear epitopes . However, evidence for the binding of 505.1 at the glutamine catalytic site was shown by using site-directed mutants of GlmS as well as by competition experiments with an irreversible inhibitor . The mAb 105.1, which recognizes the octapeptide containing the sequence RWATHG conserved among the six glucosamine-6P synthases reported so far, allowed the detection of the human enzyme. Arch Biochem Biophys, 1995 Dec 20, 324(2), 379 - 84 Cloning, sequence, and expression of mouse protoporphyrinogen oxidase; Dailey TA et al.; Protoporphyrinogen oxidase (EC 1.3.3.4) is the penultimate enzyme in the heme biosynthetic pathway, catalyzing the six-electron oxidation of protoporphyrinogen to protoporphyrin . A dominantly inherited genetic deficiency in this enzyme results in the disease variegate porphyria . We now report the cloning, sequence, and expression of mouse protoporphyrinogen oxidase . The cDNA for mouse protoporphyrinogen oxidase was obtained by complementation of Escherichia coli SASX38, a protoporphyrinogen oxidase-deficient strain, with a mouse erythroleukemia (MEL) cell expression library . The sequence of this cDNA along with 5' untranslated sequence obtained by 5' rapid amplification of cDNA ends of MEL cell mRNA is 1814 bp in length and contains an open reading frame of 1431 bp . This encodes a protein of 477 amino acid residues with a calculated molecular weight of 50,870 . The protein as expressed in E . coli is sensitive to inhibition by the diphenyl ether herbicide acifluorfen . Northern blot analyses of RNA from uninduced and induced MEL cells as well as mouse hepatoma cells all show two major mRNA species of 1.8 and 3.6 kb. Virology, 1995 Dec 20, 214(2), 439 - 44 Avian sarcoma leukemia virus protease linked to the adjacent Gag polyprotein is enzymatically active; Arad G et al.; The activity of avian sarcoma leukemia virus (ASLV) protease (PR) prior to its release from the precursor protein was determined by introducing mutations at the cleavage site between PR and the adjacent upstream nucleocapsid (NC) protein . Gag DNA fragments containing these mutations were cloned into expression vectors and introduced into Escherichia coli in which the ASLV proteins were expressed . The dipeptide NC-PR containing these mutations did not undergo autoprocessing when expressed in bacterial cells and the fused proteins were devoid of enzymatic activity . However, when the whole Gag polyprotein containing these mutations was expressed in bacterial cells, other PR cleavage sites in the viral Gag polyprotein underwent normal cleavage, indicating that the release of free PR is not a prerequisite for correct processing of the ASLV Gag precursor. Mol Gen Genet, 1995 Dec 20, 249(6), 591 - 9 Mutation frequency decline in Escherichia coli . II . Kinetics support the involvement of transcription-coupled excision repair; Bockrath R et al.; Mutation frequency decline (MFD) in Escherichia coli was examined to demonstrate repair of targeting photoproducts during the post-UV incubation required in this process . Repair of mutation-targeting cyclobutane pyrimidine dimers (T < > C) was demonstrated when a correlation was established between the mutation frequency normally associated with these lesions and the rate of mutation production at these lesions by spontaneous deamination of cytosines and photoreversal in ung-defective cells . An incubation producing a decline in mutation frequency, i.e., MFD, also produces lower rates of mutation increase via the deamination mechanism . Since the latter assay involves processes entirely within the post-UV incubation period, the lower rates are attributed to rapid transcription-coupled nucleotide excision repair (TCR) that reduces the number of relevant T < > C dimers during this period . Rediscovery of the neglected fact that MFD can be stimulated by post-UV incubation in buffer alone is part of the analysis . Results presented here and a variety of others are discussed to support a model of MFD as a particular example of TCR: effective repair of photoproducts in the transcribed DNA strand that target glutamine tRNA suppressor mutations occurs during the appropriate post-UV incubation and is responsible for MFD. Mol Gen Genet, 1995 Dec 20, 249(6), 585 - 90 Mutation frequency decline in Escherichia coli . I . Effects of defects in mismatch repair; Li BH et al.; Mutation frequency decline (MFD) in Escherichia coli was examined for effects associated with genetic defects in mismatch repair . The kinetics of MFD are slower when the B/r strain WU3610 carries the mutation mutS201::Tn5 or mutL::Tn10, both of which affect mismatch repair . Similar slow kinetics are produced by mutH34 but not by mutH471::Tn5; the latter has no apparent effect . Strain WU3610-45 (mfd-1) produces the slower kinetics if transcription is inhibited during the post-UV incubation, although it produces no decline in normal circumstances . The slower kinetics are therefore attributed to bulk excision repair that remains when rapid transcription-coupled repair (TCR) is eliminated by certain defects in mismatch repair . A model is proposed wherein mismatch repair defects are thought to slow the activity of TCR but, unlike an mfd defect, not to impede dissociation of stalled transcription complexes at lesions in the transcribed DNA strand. J Natl Cancer Inst, 1995 Dec 20, 87(24), 1853 - 61 Induction of T-cell immunity against Ras oncoproteins by soluble protein or Ras-expressing Escherichia coli; Fenton RG et al.; BACKGROUND: Point mutations in the ras proto-oncogene that activate its oncogenic potential occur in approximately 30% of human cancers . Previous studies have demonstrated that T-cell immunity against some forms of mutant Ras proteins could be elicited, and some effectiveness against tumors expressing activated Ras has been reported . PURPOSE: The goal of this study was to determine if immunization of mice with two forms of mutant Ras protein can induce high levels of Ras mutation-specific T-cell immunity in vitro and tumor regression in vivo . METHODS: Mice (BALB/c or C3H/HeJ) were immunized subcutaneously at 2-week intervals with purified Ras oncoproteins mixed with the immunologic adjuvants Antigen Formulation or QS-21, both of which have been shown to enhance the induction of T-cell-mediated immunity when included as components of soluble protein vaccines . In some experiments, mice were immunized directly with heat-killed Escherichia coli that had been induced to express one of the mutant Ras proteins . Spleen cells plus lymph node cells from Ras-immunized mice were tested in vitro for lysis of syngeneic Ras-expressing tumor cells and proliferation in response to mutant Ras peptides . For some of the cytolytic activity experiments, the spleen cells were grown under TH1 conditions (growth in presence of interleukin 2, interferon gamma, and an antibody directed against interleukin 4 to stimulate a cell-mediated immune response) or TH2 conditions (growth in presence of interleukins 2 and 4 to stimulate a humoral immune response) . The specificity of immunity was examined in vivo by challenge of Ras-immunized mice with syngeneic tumor cells expressing mutant Ras oncoproteins (HaBalb, i.e., BALB/c mouse cells expressing Ras with arginine substituted at amino acid position 12 {Arg 12 Ras}; C3HL61, i.e., C3H/HeJ mouse cells expressing Ras with leucine substituted at position 61 {Leu 61 Ras}) . Ten mice per group were used in each experiment . RESULTS: Proliferative and cytolytic T-cell responses directed against the Arg 12 Ras protein were generated in BALB/c mice, resulting in protection against challenge with cells expressing Arg 12 Ras and therapeutic benefit in mice bearing established tumors expressing this protein . In C3H/HeJ mice, high levels of cytolytic and proliferative responses were induced against Leu 61 Ras . Immunization with heat-killed E . coli genetically engineered to express Leu 61 Ras also led to the induction of anti-Ras T-cell immunity . T cells grown under TH1 conditions were cytolytic against Ras-transformed tumor cells, whereas those grown under TH2 conditions were not . CONCLUSIONS: Immunization as described here leads to Ras mutation-specific antitumor immunity in vitro and in vivo, with therapeutic efficacy in an established tumor model. Biochemistry, 1995 Dec 19, 34(50), 16359 - 64 Effect of hemimethylation and methylation of adenine on the structure and stability of model DNA duplexes; Guo Q et al.; Enzymatic methylation of adenine underlies a variety of biological regulatory mechanisms in Escherichia coli . We present here structural and thermodynamic characterization of a non-self-complementary DNA decamer duplex containing the dam sequence 5'-GATC in the unmethylated, hemimethylated (both forms), and methylated states . Differential scanning calorimetry measurements show that the free energies for adenine methylation of the decamer duplex are +1.1 and +2.0 kcal/mol for hemimethylation, respectively, and +3.3 kcal/mol for full methylation . In all cases, a large unfavorable enthalpy change is partially compensated by a favorable entropy term . CD spectroscopy indicates an overall conformational difference between the unmethylated decamer duplex and its methylated analogs . Reaction with diethyl pyrocarbonate (DEPC), a purine-specific probe sensitive to conformation, is enhanced in the vicinity of the methylation site of the duplex, consistent with loosening of base pairing at this site . Comparison of the scission patterns of these decamer duplexes by the reactive probes methidiumpropyl-EDTA.FeII {MPE.FeII} and CuI(o-phenanthroline)2 {(OP)2CuI} indicates that the methylation site of the decamer duplex represents a site of enhanced reactivity for these agents . On the basis of these thermodynamics and structural features, we suggest that the methylated base pair exists in two different helical states, which require local transient opening of the duplex for interconversion. Proc Natl Acad Sci U S A, 1995 Dec 19, 92(26), 12309 - 13 UGA suppression by a mutant RNA of the large ribosomal subunit; Jemiolo DK et al.; A role for rRNA in peptide chain termination was indicated several years ago by isolation of a 168 rRNA (small subunit) mutant of Escherichia coli that suppressed UGA mutations . In this paper, we describe another interesting rRNA mutant, selected as a translational suppressor of the chain-terminating mutant trpA (UGA211) of E . coli . The finding that it suppresses UGA at two positions in trpA and does not suppress the other two termination codons, UAA and UAG, at the same codon positions (or several missense mutations, including UGG, available at one of the two positions) suggests a defect in UGA-specific termination . The suppressor mutation was mapped by plasmid fragment exchanges and in vivo suppression to domain II of the 23S rRNA gene of the rrnB operon . Sequence analysis revealed a single base change of G to A at residue 1093, an almost universally conserved base in a highly conserved region known to have specific interactions with ribosomal proteins, elongation factor G, tRNA in the A-site, and the peptidyltransferase region of 23S rRNA . Several avenues of action of the suppressor mutation are suggested, including altered interactions with release factors, ribosomal protein L11, or 16S rRNA . Regardless of the mechanism, the results indicate that a particular residue in 23S rRNA affects peptide chain termination, specifically in decoding of the UGA termination codon. Proc Natl Acad Sci U S A, 1995 Dec 19, 92(26), 12300 - 3 Distance determination in proteins using designed metal ion binding sites and site-directed spin labeling: application to the lactose permease of Escherichia coli; Voss J et al.; As shown in the accompanying paper, the magnetic dipolar interaction between site-directed metal-nitroxide pairs can be exploited to measure distances in T4 lysozyme, a protein of known structure . To evaluate this potentially powerful method for general use, particularly with membrane proteins that are difficult to crystallize, both a paramagnetic metal ion binding site and a nitroxide side chain were introduced at selected positions in the lactose permease of Escherichia coli, a paradigm for polytopic membrane proteins . Thus, three individual cysteine residues were introduced into putative helix IV of a lactose permease mutant devoid of native cysteine residues containing a high-affinity divalent metal ion binding site in the form of six contiguous histidine residues in the periplasmic loop between helices III and IV . In addition, the construct contained a biotin acceptor domain in the middle cytoplasmic loop to facilitate purification . After purification and spin labeling, electron paramagnetic resonance spectra were obtained with the purified proteins in the absence and presence of Cu(II) . The results demonstrate that positions 103, 111, and 121 are 8, 14, and > 23 A from the metal binding site . These data are consistent with an alpha-helical conformation of transmembrane domain IV of the permease . Application of the technique to determine helix packing in lactose permease is discussed. Proc Natl Acad Sci U S A, 1995 Dec 19, 92(26), 12225 - 9 Purification, cDNA cloning, functional expression, and characterization of a 26-kDa endogenous mammalian carboxypeptidase inhibitor; Normant E et al.; The recent demonstration of the occurrence in rat brain and other nonpancreatic tissues of carboxypeptidase A (CPA) gene transcripts without associated catalytic activity could be ascribed to the presence of a soluble endogenous protein inhibitor . This tissue carboxypeptidase inhibitor (TCI), detected by the inhibition of added bovine pancreatic CPA, was purified from rat brain . Peptides were obtained by partial proteolysis of purified TCI, a protein of approximately 30 kDa, and starting from their sequences, a full-length cDNA encoding a 223-amino acid protein containing three potential phosphorylation sites was cloned from a cDNA library . Its identity with TCI was shown by expression in Escherichia coli of a recombinant protein recognized by antibodies raised against native TCI and display characteristic CPA-inhibiting activity . TCI appears as a hardly reversible, non-competitive, and potent inhibitor of CPA1 and CPA2 (Ki approximately 3 nM) and mast-cell CPA (Ki = 16 nM) and inactive on various other proteases . This pattern of selectivity might be attributable to a limited homology of a 11-amino acid sequence with sequences within the activation segments of CPA and CPB known to interact with residues within their active sites . The widespread expression of TCI in a number of tissues (e.g., brain, lung, or digestive tract) and its apparently cytosolic localization point to a rather general functional role, e.g., in the control of cytosolic protein degradation. Proc Natl Acad Sci U S A, 1995 Dec 19, 92(26), 12205 - 9 A soluble domain of the membrane-anchoring chain of influenza virus hemagglutinin (HA2) folds in Escherichia coli into the low-pH-induced conformation; Chen J et al.; The extensive refolding of the membrane-anchoring chain of hemagglutinin (HA) of influenza virus (termed HA2) in cellular endosomes, which initiates viral entry by membrane fusion, suggests that viral HA is meta-stable . HA2 polypeptide residues 38-175 expressed in Escherichia coli are reported here to fold in vivo into a soluble trimer . The structure appears to be the same as the low-pH-induced conformation of viral HA2 by alpha-helical content, thermodynamic stability, protease dissection, electron microscopy, and antibody binding . These results provide evidence that the structure of the low-pH-induced fold of viral HA2 (TBHA2) observed crystallographically is the lowest-energy-state fold of the HA2 polypeptide . They indicate that the HA2 conformation in viral HA before low pH activation of its fusion potential is metastable and suggest that removal of the receptor-binding chain (HA1) is enough to allow HA2 to adopt the stable state . Further, they provide direct evidence that low pH is not required to form the membrane-fusion conformation but acts to make this state kinetically accessible in viral HA. Proc Natl Acad Sci U S A, 1995 Dec 19, 92(26), 12120 - 5 Active site topology and reaction mechanism of GTP cyclohydrolase I; Nar H et al.; GTP cyclohydrolase I of Escherichia coli is a torus-shaped homodecamer with D5 symmetry and catalyzes a complex ring expansion reaction conducive to the formation of dihydroneopterin triphosphate from GTP . The x-ray structure of a complex of the enzyme with the substrate analog, dGTP, bound at the active site was determined at a resolution of 3 A . In the decamer, 10 equivalent active sites are present, each of which contains a 10-A deep pocket formed by surface areas of 3 adjacent subunits . The substrate forms a complex hydrogen bond network with the protein . Active site residues were modified by site-directed mutagenesis, and enzyme activities of the mutant proteins were measured . On this basis, a mechanism of the enzyme-catalyzed reaction is proposed . Cleavage of the imidazole ring is initiated by protonation of N7 by His-179 followed by the attack of water at C8 of the purine system . Cystine Cys-110 Cys-181 may be involved in this reaction step . Opening of the imidazole ring may be in concert with cleavage of the furanose ring to generate a Schiff's base from the glycoside . The gamma-phosphate of GTP may be involved in the subsequent Amadori rearrangement of the carbohydrate side chain by activating the hydroxyl group of Ser-135. Proc Natl Acad Sci U S A, 1995 Dec 19, 92(26), 12017 - 20 Adaptive mutation sequences reproduced by mismatch repair deficiency; Longerich S et al.; Adaptive reversions of a lac frameshift mutation in Escherichia coli are -1 deletions in small mononucleotide repeats, whereas growth-dependent reversions are heterogeneous . The adaptive mutations resemble instability of simple repeats, which, in hereditary colon cancer, in yeast, and in E . coli occurs in the absence of mismatch repair . The postulate that mismatch repair is disabled transiently during adaptive mutation in E . coli is supported here by the demonstration that the growth-dependent mutation spectrum can be made indistinguishable from adaptive mutations by disallowing mismatch repair during growth . Physiologically induced mismatch repair deficiency could be an important mutagenic mechanism in cancers and in evolution. Proc Natl Acad Sci U S A, 1995 Dec 19, 92(26), 11955 - 9 Crystal structure of the membrane-exposed domain from a respiratory quinol oxidase complex with an engineered dinuclear copper center; Wilmanns M et al.; Cytochrome oxidase is a membrane protein complex that catalyzes reduction of molecular oxygen to water and utilizes the free energy of this reaction to generate a transmembrane proton gradient during respiration . The electron entry site in subunit II is a mixed-valence dinuclear copper center in enzymes that oxidize cytochrome c . This center has been lost during the evolution of the quinoloxidizing branch of cytochrome oxidases but can be restored by engineering . Herein we describe the crystal structures of the periplasmic fragment from the wild-type subunit II (CyoA) of Escherichia coli quinol oxidase at 2.5-A resolution and of the mutant with the engineered dinuclear copper center (purple CyoA) at 2.3-A resolution . CyoA is folded as an 11-stranded mostly antiparallel beta-sandwich followed by three alpha-helices . The dinuclear copper center is located at the loops between strands beta 5-beta 6 and beta 9-beta 10 . The two coppers are at a 2.5-A distance and symmetrically coordinated to the main ligands that are two bridging cysteines and two terminal histidines . The residues that are distinct in cytochrome c and quinol oxidases are around the dinuclear copper center . Structural comparison suggests a common ancestry for subunit II of cytochrome oxidase and blue copper-binding proteins. FEBS Lett, 1995 Dec 18, 377(2), 249 - 52 Human ClpP protease: cDNA sequence, tissue-specific expression and chromosomal assignment of the gene; Bross P et al.; We identified three overlapping human expressed sequence tags with significant homology to the E . coli ClpP amino sequence by screening the EMBL nucleotide database . With this sequence information we applied 5' and 3'-rapid amplification of cDNA ends (RACE) to amplify and sequence human clpP cDNA in two overlapping fragments . The open reading frame encodes a 277 amino acid long precursor polypeptide . Two ClpP specific motifs surrounding the active site residues are present and extensive homology to ClpP's from other organisms was observed . Northern blotting showed high relative expression levels of clpP mRNA in skeletal muscle, intermediate levels in heart, liver and pancreas, and low levels in brain, placenta, lung and kidney . By analysis of human/rodent cell hybrids the human clpP gene was assigned to chromosome 19. FEBS Lett, 1995 Dec 18, 377(2), 221 - 6 A novel approach for expression cloning of small GTPases: identification, tissue distribution and chromosome mapping of the human homolog of rheb; Gromov PS et al.; We report a novel approach for identifying monomeric GTP-binding proteins that is based on probing cDNA expression libraries with {alpha-32P}GTP . In short, a nitrocellulose replica from a plated cDNA expression library is treated with 2% SDS to block the GTP-binding activity of various G proteins expressed by E . coli, thus allowing the direct identification of positive clones . Using this procedure we have cloned several small GTP-binding proteins from human keratinocytes including the human homolog of rheb, a novel member of the ras-related GTP-binding proteins . Human rheb cDNA shares 90% identity with the rat counterpart and it is highly upregulated in transformed human cells of various origin . Northern analysis showed that human rheb is ubiquitously expressed, with the highest levels observed in skeletal and cardiac muscle, and not in brain, as it is the case for rat rheb . The human RHEB gene was mapped to chromosome 10q11. FEBS Lett, 1995 Dec 18, 377(2), 193 - 6 Characterization of functionally independent domains in the human ubiquitin conjugating enzyme UbcH2; Kaiser P et al.; UbcH2 encodes a human ubiquitin conjugating enzyme (E2) able to conjugate ubiquitin to histone H2A in an E3 independent manner in vitro, which indicates that UbcH2 directly interacts with its substrates . To identify parts of the enzyme that are capable of binding H2A, we expressed several deletion mutants of UbcH2 in E . coli and tested the ability of the affinity purified mutant proteins to ubiquitinate H2A in the presence of bacterial expressed E1 and ubiquitin . With this in vitro assay we identified a C-terminal part of UbcH2 to be important for the interaction with H2A . Transfer of this C-terminal domain to another human E2, which is unable to catalyze ubiquitination of histones, leads to a fully active hybrid human ubiquitin conjugating enzyme capable of H2A ubiquitination . These results demonstrate that UbcH2 consists of two functionally independent domains . A N-terminal core domain with ubiquitin conjugating activity, and a C-terminal domain which interacts with substrate proteins. FEBS Lett, 1995 Dec 18, 377(2), 172 - 4 Synthesis, cloning and expression in Escherichia coli of a gene coding for the Met8-->Leu CMTI I--a representative of the squash inhibitors of serine proteinases; Bolewska K et al.; A chemically synthesized gene coding for a Cucurbita maxima trypsin inhibitor modified at position P'3 (Met8-->Leu CMTI I), i.e . at the third position downstream of the reactive site bond (Arg5-Ile), was cloned into a derivative of the plasmid pAED4 that utilizes a T7 expression system . The gene was expressed in Escherichia coli as a fusion protein that accumulates in inclusion bodies . After reduction and CNBr cleavage of the fusion protein followed by oxidative refolding and reverse-phase HPLC, about 5 mg of pure protein was obtained per 1 of cell culture . Association constants of recombinant Leu-8-CMTI I with bovine beta-trypsin and human cathepsin G are the same, within experimental error, as for CMTI I isolated from a natural source. FEBS Lett, 1995 Dec 18, 377(2), 155 - 8 Precise mapping of the tms1 binding site on p53; Wagner P et al.; Originally identified as multicopy suppressor of a lethal growth arrest caused by expression of a tumour mutant cDNA of p53 in fission yeast the tms1 gene product was found to form stable complexes with p53 in yeast . By using purified recombinant proteins multimeric complexes of tms1 and p53 could be demonstrated and recently the p53 binding site on the tms1 protein was established to the sequence YYITTEDFCT (aa 116-125) in the vicinity of a well conserved cell division motif . Here we report the precise mapping of the tms1 binding site on the p53 protein to the sequence LQIRGRERFE (aa 330-339) which defines a new functional domain on the p53 protein. FEBS Lett, 1995 Dec 18, 377(2), 123 - 7 Molecular cloning and functional expression of a recombinant 72.5 kDa fragment of the 110 kDa regulatory subunit of smooth muscle protein phosphatase 1M; Haystead CM et al.; We have cloned a partial rat kidney cDNA that encodes a 72.5 kDa N terminal fragment of a third isoform of the M110 subunit of phosphatase 1 . This new isoform contains an insert in the 542-597 position not present in the M110 previously cloned (Chen et al . (1994) FEBS Lett . 356, 51-55) from the same species . The encoded cDNA was expressed as a soluble GST-fusion protein in E . coli, and its ability to interact with native PP-1C was measured both in vitro and in permeabilized smooth muscle . In vitro, the fusion protein was capable of selectively binding PP-1C and increasing the substrate specificity of the phosphatase towards myosin 13.2 +/- 3.5-fold (S.E . of the mean, n = 3) . In permeabilized smooth muscle pretreated with microcystin, the recombinant protein alone (1.0 microM) did not cause relaxation, but did significantly enhance the ability of PP-1C (0.3 microM) to relax the muscle . These findings show that the N terminal domain of the M110 subunit is the primary site for both PP-1C and myosin binding, and thereby determines myosin specificity . The presence of isoformic variation within this sequence may permit organ/cell specific regulation of phosphorylation sites. Structure, 1995 Dec 15, 3(12), 1407 - 19 Structure of the biotinyl domain of acetyl-coenzyme A carboxylase determined by MAD phasing; Athappilly FK et al.; BACKGROUND: Acetyl-coenzyme A carboxylase catalyzes the first committed step of fatty acid biosynthesis . Universally, this reaction involves three functional components all related to a carboxybiotinyl intermediate . A biotinyl domain shuttles its covalently attached biotin prosthetic group between the active sites of a biotin carboxylase and a carboxyl transferase . In Escherichia coli, the three components reside in separate subunits: a biotinyl domain is the functional portion of one of these, biotin carboxy carrier protein (BCCP) . RESULTS: We have expressed natural and selenomethionyl (Se-met) BCCP from E . coli as biotinylated recombinant proteins, proteolyzed them with subtilisin Carlsberg to produce the biotinyl domains BCCP and Se-met BCCPsc, determined the crystal structure of Se-met BCCPsc using a modified version of the multiwavelength anomalous diffraction (MAD) phasing protocol, and refined the structure for the natural BCCPsc at 1.8 A resolution . The structure may be described as a capped beta sandwich with quasi-dyad symmetry . Each half contains a characteristic hammerhead motif . The biotinylated lysin is located at a hairpin beta turn which connects the two symmetric halves of the molecule, and its biotinyl group interacts with a non-symmetric protrusion from the core . CONCLUSIONS: This first crystal structure of a biotinyl domain helps to unravel the central role of such domains in reactions catalyzed by biotin-dependent carboxylases . The hammerhead structure observed twice in BCCPsc may be regarded as the basic structural motif of biotinyl and lipoyl domains of a superfamily of enzymes . The new MAD phasing techniques developed in the course of determining this structure enhance the power of the MAD method. Structure, 1995 Dec 15, 3(12), 1323 - 32 Structure and catalytic mechanism of glucosamine 6-phosphate deaminase from Escherichia coli at 2.1 A resolution; Oliva G et al.; BACKGROUND: Glucosamine 6-phosphate deaminase from Escherichia coli is an allosteric hexameric enzyme which catalyzes the reversible conversion of D-glucosamine 6-phosphate into D-fructose 6-phosphate and ammonium ion and is activated by N-acetyl-D-glucosamine 6-phosphate . Mechanistically, it belongs to the group of aldoseketose isomerases, but its reaction also accomplishes a simultaneous amination/deamination . The determination of the structure of this protein provides fundamental knowledge for understanding its mode of action and the nature of allosteric conformational changes that regulate its function . RESULTS: The crystal structure of glucosamine 6-phosphate deaminase with bound phosphate ions is presented at 2.1 A resolution together with the refined structures of the enzyme in complexes with its allosteric activator and with a competitive inhibitor . The protein fold can be described as a modified NAD-binding domain . CONCLUSIONS: From the similarities between the three presented structures, it is concluded that these represent the enzymatically active R state conformer . A mechanism for the deaminase reaction is proposed . It comprises steps to open the pyranose ring of the substrate and a sequence of general base-catalyzed reactions to bring about isomerization and deamination, with Asp72 playing a key role as a proton exchanger. Structure, 1995 Dec 15, 3(12), 1307 - 14 X-ray structure of human nucleoside diphosphate kinase B complexed with GDP at 2 A resolution; Morera S et al.; BACKGROUND: Nucleoside diphosphate (NDP) kinases provide precursors for DNA and RNA synthesis . In mammals, these enzymes are also involved in cell regulations . Human NDP kinase B, product of the human nm23-H2 gene, is both an enzyme and a transcription factor . It activates transcription of the c-myc oncogene independently of its catalytic function,