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H Zalkin

Publications and source records attributed to H Zalkin.

At least 109 records · Page 6Linked to original sources
PubMed1985

Identification of a trpG-related glutamine amide transfer domain in Escherichia coli GMP synthetase.

An improved method was developed to align related protein sequences and search for homology. A glutamine amide transfer domain was identified in an NH2-terminal segment of GMP synthetase from Escherichia coli. Amino acid residues 1-198 in GMP synthetase are homologous with the glutamine amide transfer domain in trpG X D-encoded anthranilate synthase component II-anthranilate phosphoribosyltransferase and the related pabA-encoded p-aminobenzoate synthase component II. This result supports a model for gene fusion in which a trpG-related glutamine amide transfer domain was recruited to augment the function of a primitive NH3-dependent GMP synthetase. Sequence analyses emphasize that glutamine amide transfer domains are thus far found only at the NH2 terminus of fused proteins. Two rules are formulated to explain trpG and trpG-related fusions. (i) trpG and trpG-related genes must have translocated immediately up-stream of genes destined for fusion in order to position a glutamine amide transfer domain at the NH2 terminus after fusion. (ii) trpG and trpG-related genes could not translocate adjacent to a regulatory region at the 5' end of an operon. These rules explain known trpG-like fusions and explain why trpG and pabA are not fused to trpE and pabB, respectively. Alignment searches of GMP synthetase with two other enzymes that bind GMP, E. coli amidophosphoribosyltransferase and human hypoxanthine-guanine phosphoribosyltransferase, suggest a structurally homologous segment which may constitute a GMP binding site.

Amidophosphoribosyltransferase↗
PubMed1985

Study of anthranilate synthase function by replacement of cysteine 84 using site-directed mutagenesis.

Cysteine 84 was replaced by glycine in Serratia marcescens anthranilate synthase Component II using site-directed mutagenesis of cloned trpG. This replacement abolished the glutamine-dependent anthranilate synthase activity but not the NH3-dependent activity of the enzyme. The mutation provides further evidence for the role of active site cysteine 84 in the glutamine amide transfer function of anthranilate synthase Component II. By the criteria of circular dichroism, proteolytic inactivation, and feedback inhibition the mutant and wild type enzymes were structurally similar. The NH3-dependent anthranilate synthase activity of the mutant enzyme supported tryptophan synthesis in media containing a high concentration of ammonium ion.

Amino Acid Sequence↗
PubMed1985

GMP synthetase.

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Ammonia↗
PubMed1985

CTP synthetase.

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Binding Sites↗
PubMed1985

NAD synthetase.

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Amide Synthases↗
PubMed1984

Glutamine amidotransferase function. Replacement of the active-site cysteine in glutamine phosphoribosylpyrophosphate amidotransferase by site-directed mutagenesis.

Site-directed mutagenesis was employed to replace cysteine 12 with phenylalanine in Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase (amidophosphoribosyltransferase). Glutamine-dependent amidophosphoribosyltransferase activity was abolished as a consequence of the mutation. The mutant enzyme, however, exhibited NH3-dependent activity, contained Fe-S, and was normally regulated by AMP. These results document the role of the active site cysteine in activation of glutamine for amide transfer. NH3-dependent amidophosphoribosyltransferase was utilized for de novo purine nucleotide synthesis. Cells containing the mutant enzyme grew at nearly the wild-type rate in media containing a high concentration of NH4Cl. The Phe-12 mutation was used to study NH2-terminal processing. Whereas the wild-type Cys-12 enzyme is processed correctly in Escherichia coli by removal of 11 amino acid residues from the NH2 terminus, the Phe-12 mutant enzyme was not subject to undecapeptide processing. Neither the mutant nor wild-type enzyme made in vitro was correctly processed. Alternative enzymatic and autocatalytic processing mechanisms were considered. The available evidence favors autocatalytic NH2-terminal undecapeptide processing.

Adenosine Monophosphate↗
PubMed1984

Glutamine nucleotide sequence of Saccharomyces cerevisiae ADE4 encoding phosphoribosylpyrophosphate amidotransferase.

Saccharomyces cerevisiae gene ADE4 encoding glutamine phosphoribosylpyrophosphate amidotransferase (amidophosphoribosyltransferase) has been cloned by complementation of an ade4 auxotroph. The nucleotide sequence of ADE4 along with upstream and downstream flanking sequences was determined. The ADE4 structural gene consists of 1530 base pairs from which a 510-amino acid translation product, Mr = 56,691, was deduced. Yeast amidophosphoribosyltransferase is homologous to the enzymes from Escherichia coli and Bacillus subtilis. The active site cysteine residue in the bacterial amidophosphoribosyltransferases which functions in glutamine amide transfer is conserved in the yeast enzyme. Yeast amidophosphoribosyltransferase does not contain the previously deduced sequence required for binding of a [4Fe-4S] center indicating that a [4Fe-4S] center is an unlikely component of the yeast enzyme. Amidophosphoribosyltransferase was stable in growing and nongrowing cells and was not inactivated or degraded. Thus in the group, S. cerevisiae, E. coli, B. subtilis, the content of a [4Fe-4S] cluster in amidophosphoribosyltransferase correlates with a mechanism for oxygen-dependent inactivation of the enzyme. Northern blots demonstrate that ADE4 expression is transcriptionally regulated. The 5' end of the ADE4 mRNA was identified by nuclease S1 mapping.

Amidophosphoribosyltransferase↗
PubMed1984

Nucleotide sequence of Saccharomyces cerevisiae genes TRP2 and TRP3 encoding bifunctional anthranilate synthase: indole-3-glycerol phosphate synthase.

Saccharomyces cerevisiae anthranilate synthase:indole-3-glycerol phosphate synthase is a multifunctional hetero-oligomeric enzyme encoded by genes TRP2 and TRP3. TRP2, encoding anthranilate synthase Component I, was cloned by complementation of a yeast trp2 mutant. The nucleotide sequence of TRP2 as well as that of TRP3 were determined. The deduced anthranilate synthase Component I primary structure from yeast exhibits only limited similarity to that of the corresponding Escherichia coli subunit encoded by trpE. On the other hand, yeast anthranilate synthase Component II and indole-3-glycerol phosphate synthase amino acid sequences from TRP3 are clearly homologous with the corresponding sequences of the E. coli trpG and trpC polypeptide segments and thereby establish the bifunctional structure of TRP3 protein. Based on comparisons of TRP3 amino acid sequence with homologous sequences from E. coli and Neurospora crassa, an 11-amino acid residue connecting segment was identified which fuses the trpG and trpC functions of the bifunctional TRP3 protein chain. These comparisons support the conclusion that the amino acid sequence of connectors in homologous multifunctional enzymes need not be conserved. Connector function is thus not dependent on a specific sequence. Nuclease S1 mapping was used to identify mRNA 5' termini. Heterogeneous 5' termini were found for both TRP2 and TRP3 mRNA. TRP2 and TRP3 5'-flanking regions were analyzed for sequences that might function in regulation of these genes by the S. cerevisiae general amino acid control system. The 9 base pair direct repeat (Hinnebusch, A.G., and Fink, G.R. (1983) J. Biol. Chem. 258, 5238-5247) and inverted repeats were identified in the 5'-flanking sequences of TRP2 and TRP3.

Amino Acid Sequence↗
PubMed1983

The amino acid sequence of D-ribose-binding protein from Escherichia coli K12.

The amino acid sequence of the D-ribose-binding protein from Escherichia coli K12 was determined from the DNA sequence of the gene. Protein sequence analyses covering 80% of the protein were consistent with the sequence deduced from the DNA. The mature binding protein has 271 amino acid residues and shows substantial homology to D-galactose-binding protein. A signal peptide sequence of 23 or 25 residues was also deduced from the DNA sequence. It shows the characteristic features of prokaryotic signal peptides.

Amino Acid Sequence↗
PubMed1983

The glutamine-utilizing site of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase.

Reaction of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase with 6-diazo-5-oxo-L-norleucine resulted in complete loss of its ability to catalyze glutamine-dependent phosphoribosylamine formation and its glutaminase activity, whereas its ability to catalyze ammonia-dependent phosphoribosylamine formation and to hydrolyze phosphoribosylpyrophosphate was increased. The site of reaction with 6-diazo-5-oxo-L-norleucine was the NH2-terminal cysteine residue. The NH2-terminal sequence of the B. subtilis enzyme was homologous with that of the corresponding amidotransferase from Escherichia coli, for which the NH2-terminal cysteine is also essential for glutamine utilization (Tso, J. Y., Hermodson, M. A., and Zalkin, H. (1982) J. Biol. Chem. 257, 3532-3536). The fact that the metal-free E. coli amidotransferase contains a glutamine-utilizing structure that is very similar to that found in B. subtilis amidotransferase, which contains an essential [4Fe-4S] center, indicates that the iron-sulfur center probably plays no role in glutamine utilization.

Amidophosphoribosyltransferase↗
PubMed1983

Cloning of the Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase gene in Escherichia coli. Nucleotide sequence determination and properties of the plasmid-encoded enzyme.

The Bacillus subtilis gene encoding glutamine phosphoribosylpyrophosphate amidotransferase (amidophosphoribosyltransferase) was cloned in pBR322. This gene is designated purF by analogy with the corresponding gene in Escherichia coli. B. subtilis purF was expressed in E. coli from a plasmid promoter. The plasmid-encoded enzyme was functional in vivo and complemented an E. coli purF mutant strain. The nucleotide sequence of a 1651-base pair B. subtilis DNA fragment was determined, thus localizing the 1428-base pair structural gene. A primary translation product of 476 amino acid residues was deduced from the DNA sequence. Comparison with the previously determined NH2-terminal amino acid sequence indicates that 11 residues are proteolytically removed from the NH2 terminus, leaving a protein chain of 465 residues having an NH2-terminal active site cysteine residue. Plasmid-encoded B. subtilis amidophosphoribosyltransferase was purified from E. coli cells and compared to the enzymes from B. subtilis and E. coli. The plasmid-encoded enzyme was similar in properties to amidophosphoribosyltransferase obtained from B. subtilis. Enzyme specific activity, immunological reactivity, in vitro lability to O2, Fe-S content, and NH2-terminal processing were virtually identical with amidophosphoribosyltransferase purified from B. subtilis. Thus E. coli correctly processed the NH2 terminus and assembled [4Fe-4S] centers in B. subtilis amidophosphoribosyltransferase although it does not perform these maturation steps on its own enzyme. Amino acid sequence comparison indicates that the B. subtilis and E. coli enzymes are homologous. Catalytic and regulatory domains were tentatively identified based on comparison with E. coli amidophosphoribosyltransferase and other phosphoribosyltransferase (Argos, P., Hanei, M., Wilson, J., and Kelley, W. (1983) J. Biol. Chem. 258, 6450-6457).

Amidophosphoribosyltransferase↗
PubMed1983

Chemical modification and ligand binding studies with Escherichia coli glutamate synthase.

The structure and function of Escherichia coli glutamate synthase were studied by ligand binding and chemical modification experiments. Binding of NADP+ was to a single dinucleotide site per alpha beta protomer with a Kd of approximately 5 microM. Phenylglyoxal modified an essential arginyl residue required for binding of NADP+. E. coli glutamate synthase thus employs a single dinucleotide binding site which functions in the NADPH to flavin electron transfer for glutamine-dependent glutamate synthase and for direct reduction of 2-iminoglutarate by NADPH in the NH3-dependent reaction. Binding of 2-oxoglutarate was complex. "Half of the sites" binding of 2-oxoglutarate (Kd less than 0.25 microM) was obtained in the absence of glutamine. Binding to half of the sites was pH independent. In the presence of glutamine, the 2-oxoglutarate binding ratio was approximately 1 equiv per protomer (Kd = 2-3 microM) at pH 7.5. This binding was pH dependent and varied between 0.43 equiv per protomer at pH 6.7 and 2.3 equiv per protomer at pH 9.0. Correlation of half of the sites binding with negative cooperativity for 2-oxoglutarate saturation indicates the utilization of low-Kd 2-oxoglutarate sites for NH3-dependent glutamate synthase. Binding of glutamine promotes a conformational change that exposes additional 2-oxoglutarate sites having a Kd of 2-3 microM which are utilized in the glutamine-dependent reaction. Chemical modification with pyridoxal 5'-phosphate caused inactivation of glutamine-dependent but not NH3-dependent glutamate synthase. Inactivation was ascribed to modification of one to two lysyl residues per protomer by Schiff base formation. The essential lysyl residue has a role in the binding of glutamine.

Escherichia coli↗