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H Zalkin

Publications and source records attributed to H Zalkin.

At least 19 recordsLinked to original sources
PubMed1999

Mutational analysis of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase propeptide processing.

Glutamine phosphoribosylpyrophosphate amidotransferase from Bacillus subtilis is a member of an N-terminal nucleophile hydrolase enzyme superfamily, several of which undergo autocatalytic propeptide processing to generate the mature active enzyme. A series of mutations was analyzed to determine whether amino acid residues required for catalysis are also used for propeptide processing. Propeptide cleavage was strongly inhibited by replacement of the cysteine nucleophile and two residues of an oxyanion hole that are required for glutaminase function. However, significant propeptide processing was retained in a deletion mutant with multiple defects in catalysis that was devoid of enzyme activity. Intermolecular processing of noncleaved mutant enzyme subunits by active wild-type enzyme subunits was not detected in hetero-oligomers obtained from a coexpression experiment. While direct in vitro evidence for autocatalytic propeptide cleavage was not obtained, the results indicate that some but not all of the amino acid residues that have a role in catalysis are also needed for propeptide processing.

Amidophosphoribosyltransferase↗
PubMed1998

Escherichia coli purine repressor: key residues for the allosteric transition between active and inactive conformations and for interdomain signaling.

The Escherichia coli purine repressor, PurR, exists in an equilibrium between open and closed conformations. Binding of a corepressor, hypoxanthine or guanine, shifts the allosteric equilibrium in favor of the closed conformation and increases the operator DNA binding affinity by 40-fold compared to aporepressor. Glu70 and Trp147 PurR mutations were isolated which perturb the allosteric equilibrium. Three lines of evidence indicate that the allosteric equilibrium of E70A and W147A aporepressors was shifted toward the closed conformation. First, compared to wild-type PurR, these mutant repressors had a 10-30-fold higher corepressor binding affinity. Second, the mutant aporepressors bound to operator DNA with an affinity that is characteristic of the wild-type PurR holorepressor. Third, binding of guanine to wild-type PurR resulted in a near-UV circular dichroism spectral change at 297-305 nm that is attributed to the closed conformation. The circular dichroism spectrum of the E70A aporepressor at 297-305 nm was that expected for the closed conformation, and it was not appreciably altered by corepressor binding. Mutational analysis was used to identify an Arg115-Ser46' interdomain intersubunit hydrogen bond that is necessary for transmitting the allosteric transition in the corepressor binding domain to the DNA binding domain. R115A and S46G PurR mutants were defective in DNA binding in vitro and repressor function in vivo although corepressor binding was identical to the wild type. These results establish that the hydrogen bond between the side chain NH2 of Arg115 and the main chain CO of Ser46' plays a critical role in interdomain signaling.

Allosteric Regulation↗
PubMed1998

Structure-based redesign of corepressor specificity of the Escherichia coli purine repressor by substitution of residue 190.

Guanine or hypoxanthine, physiological corepressors of the Escherichia coli purine repressor (PurR), promote formation of the ternary PurR-corepressor-operator DNA complex that functions to repress pur operon gene expression. Structure-based predictions on the importance of Arg190 in determining 6-oxopurine specificity and corepressor binding affinity were tested by mutagenesis, analysis of in vivo function, and in vitro corepressor binding measurements. Replacements of Arg190 with Ala or Gln resulted in functional repressors in which binding of guanine and hypoxanthine was retained but specificity was relaxed to permit binding of adenine. X-ray structures were determined for ternary complexes of mutant repressors with purines (adenine, guanine, hypoxanthine, and 6-methylpurine) and operator DNA. These structures indicate that R190A binds guanine, hypoxanthine, and adenine with nearly equal, albeit reduced, affinity in large part because of a newly made compensatory hydrogen bond between the rotated hydroxyl side chain of Ser124 and the exocyclic 6 positions of the purines. Through direct and water-mediated contacts, the R190Q protein binds adenine with a nearly 75-fold higher affinity than the wild type repressor while maintaining wild type affinity for guanine and hypoxanthine. The results establish at the atomic level the basis for the critical role of Arg190 in the recognition of the exocyclic 6 position of its purine corepressors and the successful redesign of corepressor specificity.

Adenine↗
PubMed1998

Enzymes utilizing glutamine as an amide donor.

Amide nitrogen from glutamine is a major source of nitrogen atoms incorporated biosynthetically into other amino acids, purine and pyrimidine bases, amino-sugars, and coenzymes. A family comprised of at least sixteen amidotransferases are known to catalyze amide nitrogen transfer from glutamine to their acceptor substrates. Recent fine structural advances, largely as a result of X-ray crystallography, now provide structure-based mechanisms that help to explain fundamental aspects of the catalytic and regulatory interactions of several of these aminotransferases. This chapter provides an overview of this recent progress made on the characterization of amidotransferase structure and mechanism.

Allosteric Regulation↗
PubMed1998

Crystal structure of glutamine phosphoribosylpyrophosphate amidotransferase from Escherichia coli.

Crystal structures of glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase from Escherichia coli have been determined to 2.0-A resolution in the absence of ligands, and to 2.5-A resolution with the feedback inhibitor AMP bound to the PRPP catalytic site. Glutamine PRPP amidotransferase (GPATase) employs separate catalytic domains to abstract nitrogen from the amide of glutamine and to transfer nitrogen to the acceptor substrate PRPP. The unliganded and AMP-bound structures, which are essentially identical, are interpreted as the inhibited form of the enzyme because the two active sites are disconnected and the PRPP active site is solvent exposed. The structures were compared with a previously reported 3.0-A structure of the homologous Bacillus subtilis enzyme (Smith JL et al., 1994, Science 264:1427-1433). The comparison indicates a pattern of conservation of peptide structures involved with catalysis and variability in enzyme regulatory functions. Control of glutaminase activity, communication between the active sites, and regulation by feedback inhibitors are addressed differently by E. coli and B. subtilis GPATases. The E. coli enzyme is a prototype for the metal-free GPATases, whereas the B. subtilis enzyme represents the metal-containing enzymes. The structure of the E. coli enzyme suggests that a common ancestor of the two enzyme subfamilies may have included an Fe-S cluster.

Adenosine Monophosphate↗
PubMed1998

The structure of PurR mutant L54M shows an alternative route to DNA kinking.

The crystal structure of the purine repressor mutant L54M bound to hypoxanthine and to the purF operator provides a stereochemical understanding of the high DNA affinity of this hinge helix mutant. Comparison of the PurR L54M-DNA complex to that of the wild type PurR-DNA complex reveals that these purine repressors bind and kink DNA similarly despite significant differences in their minor groove contacts and routes to interdigitation of the central C.G:G.C base pair step. Modeling studies, supported by genetic and biochemical data, show that the stereochemistry of the backbone atoms of the abutting hinge helices combined with the rigidity of the kinked base pair step constrain the interdigitating residue to leucine or methionine for the LacI/GalR family of transcription regulators.

Amino Acid Substitution↗
PubMed1997

Coupled formation of an amidotransferase interdomain ammonia channel and a phosphoribosyltransferase active site.

Activation of gluatmine phosphoribosylpyrophosphate (RPPP) amidotransferase (GPATase) by binding of a PRPP substrate analog results in the formation of a 20 A channel connecting the active site for glutamine hydrolysis in one domain with the PRPP site in a second domain. This solvent-inaccessible channel permits transfer of the NH3 intermediate between the two active sites. Tunneling of NH3 may be a common mechanism for glutamine amidotransferase-catalyzed nitrogen transfer and for coordination of catalysis at two distinct active sites in complex enzymes. The 2.4 A crystal structure of the active conformer of GPATase also provides the first description of an intact active site for the phosphoribosyltransferase (PRTase) family of nucleotide synthesis and salvage enzymes. Chemical assistance to catalysis is provided primarily by the substrate and secondarily by the enzyme in the proposed structure-based mechanism. Different catalytic and inhibitory modes of divalent cation binding to the PRTase active site are revealed in the active conformer of the enzyme and in a feedback-inhibited GMP complex.

Amidophosphoribosyltransferase↗
PubMed1997

The X-ray structure of the PurR-guanine-purF operator complex reveals the contributions of complementary electrostatic surfaces and a water-mediated hydrogen bond to corepressor specificity and binding affinity.

The purine repressor, PurR, is the master regulatory protein of de novo purine nucleotide biosynthesis in Escherichia coli. This dimeric transcription factor is activated to bind to cognate DNA operator sites by initially binding either of its physiologically relevant, high affinity corepressors, hypoxanthine (Kd = 9.3 microM) or guanine (Kd = 1.5 microM). Here, we report the 2.5-A crystal structure of the PurR-guanine-purF operator ternary complex and complete the atomic description of 6-oxopurine-induced repression by PurR. As anticipated, the structure of the PurR-guanine-purF operator complex is isomorphous to the PurR-hypoxanthine-purF operator complex, and their protein-DNA and protein-corepressor interactions are nearly identical. The former finding confirms the use of an identical allosteric DNA-binding mechanism whereby corepressor binding 40 A from the DNA-binding domain juxtaposes the hinge regions of each monomer, thus favoring the formation and insertion of the critical minor groove-binding hinge helices. Strikingly, the higher binding affinity of guanine for PurR and the ability of PurR to discriminate against 2-oxopurines do not result from direct protein-ligand interactions, but rather from a water-mediated contact with the exocyclic N-2 of guanine, which dictates the presence of a donor group on the corepressor, and the better electrostatic complementarity of the guanine base and the corepressor-binding pocket.

Bacterial Proteins↗
PubMed1997

Mechanism of the synergistic end-product regulation of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase by nucleotides.

De novo purine nucleotide synthesis is regulated, at least in part, by end-product inhibition of glutamine PRPP amidotransferase. An important feature of this inhibition is the fact that certain synergistic nucleotide pairs give more than additive inhibition. The physiological importance of synergism is in amplifying regulation by the adenine and guanine nucleotide end products of de novo synthesis. Using a new method to quantitate synergism, ADP plus GMP were confirmed [Meyer, E., and Switzer, R. L. (1978) J. Biol. Chem. 254, 5397-5402] to give strong synergistic inhibition of Bacillus subtilis glutamine PRPP amidotransferase. An X-ray structure of the ternary enzyme.ADP.GMP complex established that ADP binds to the allosteric A site and GMP to the catalytic C site. GMP increased the binding affinity of ADP for the A site by approximately 20-fold. Synergism results from a specific nucleotide-nucleotide interaction that is dependent upon a nucleoside diphosphate in the A site and a nucleoside monophosphate in the C site. Furthermore, synergism is enhanced by the competition between nucleotide inhibitor and PRPP substrate for the C site. Purine base specificity results from a backbone carbonyl interaction of Lys305' with the 6-NH2 group of adenine in the A site and a Ser347 Ogamma interaction with the 2-NH2 group of guanine in the C site. Steric considerations favor binding of the nucleoside diphosphate to the A site. Site-directed replacements of key residues increased the nucleotide concentrations needed for 50% inhibition and in some cases perturbed synergism. Mutations in either of the nucleotide sites perturbed function at both sites, supporting the important role of synergism.

Adenosine Diphosphate↗
PubMed1997

Role of NRF-1 in bidirectional transcription of the human GPAT-AIRC purine biosynthesis locus.

GPAT and AIRC encode enzymes for steps one and six plus seven respectively in the pathway for de novo purine nucleotide synthesis in vertebrates. The human GPAT and AIRC genes are divergently transcribed from a 558 bp intergenic promoter region. Cis-acting sites and transcription factors important for bidirectional expression were identified. A cluster of sites between nt 215 and 260 are essential, although not sufficient, for expression of both genes. Two proteins from HepG2 cell nuclear extract, identified as NRF-1 and Sp1, bound to the promoter at sites within the 215-260 region. NRF-1 was required for stable binding of Sp1. Deletion of a 5'promoter region including nt 215-260 resulted in decreased expression of GPAT and AIRC in transfected HepG2 cells. The decreased expression was accounted for by point mutations in an NRF-1 site and either of two flanking sites for Sp1. These transcription factors account in part for the coordinated expression of human GPAT and AIRC.

Amidophosphoribosyltransferase↗
PubMed1997

Interaction of Bacillus subtilis purine repressor with DNA.

A purine repressor (PurR) mediates adenine nucleotide-dependent regulation of transcription initiation of the Bacillus subtilis pur operon. This repressor has been purified for the first time, and binding to control site DNA was characterized. PurR binds in vitro to four operons. Apparent Kd values for binding were 7 nM for the pur operon, 8 nM for purA, 13 nM for purR, and 44 nM for the pyr operon. In each case, DNase I footprints exhibited a pattern of protected and hypersensitive sites that extended over more than 60 bp. A GAAC-N24-GTTC sequence in the pur operon was necessary but not sufficient for the PurR-DNA interaction. However, this motif, which is conserved in the four binding sites, was not required for binding of PurR to purA. Thus, the common DNA recognition element for binding of PurR to the four operons is not known. Multiple PurR-pur operon DNA complexes having a binding stoichiometry that was either approximately two or six repressor molecules per DNA fragment were detected. The results of a torsional constraint experiment suggest that control site DNA forms one right-handed turn around PurR.

Bacillus subtilis↗
PubMed1997

Role of NifS in maturation of glutamine phosphoribosylpyrophosphate amidotransferase.

Glutamine phosphoribosylpyrophosphate amidotransferase from Bacillus subtilis is synthesized as an inactive precursor that requires two maturation steps: incorporation of a [4Fe-4S] center and cleavage of an 11-residue NH2-terminal propeptide. Overproduction from a multicopy plasmid in Escherichia coli leads to the formation of soluble proenzyme and mature enzyme forms as well as a small fraction of insoluble proenzyme. Heterologous expression of Azotobacter vinelandii nifS from a compatible plasmid increased the maturation of the soluble proenzyme three- to fourfold without influencing the content of the insoluble fraction. These results support a role for NifS in heterologous Fe-S cluster assembly and enzyme maturation.

Amidophosphoribosyltransferase↗
PubMed1996

Structure and function of the glutamine phosphoribosylpyrophosphate amidotransferase glutamine site and communication with the phosphoribosylpyrophosphate site.

Glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase from Escherichia coli exhibits a basal PRPP-independent glutaminase activity having a kcat/Km that is 0.3% of fully active enzyme. Binding of PRPP activates the enzyme by a structural change that lowers the Km for glutamine 100-fold and couples glutamine hydrolysis to synthesis of 5-phosphoribosylamine. By analysis of the x-ray structure of the glutamine site containing bound 6-diazo-5-oxonorleucine, a glutamine affinity analog, and by site-directed mutagenesis we have identified residues important for glutamine binding, catalysis, and coupling with PRPP. Tyr74 is a key residue in the coupling between the sites for glutamine in the NH2-terminal domain and PRPP in the COOH-terminal domain. Arg73 and Asp127 have roles in glutamine binding. The x-ray structure indicates that there are no amino acid side chains sufficiently close to Cys1 to participate as a proton acceptor in formation of the thiolate needed for nucleophilic attack on the carboxamide of glutamine, nor as a general acid for amide nitrogen transfer. Based on the x-ray model of the glutamine site and analysis of a mutant enzyme we propose that the free NH2 terminus of Cys1 functions as the proton acceptor and donor. The results indicate that the side chain of Asn101 and the backbone nitrogen of Gly102 function to stabilize a tetrahedral oxyanion resulting from attack of Cys1 on the glutamine carboxamide. Cys1, Arg73, Asn101, Gly102, and Asp127 are conserved in the NH2-terminal domain of a subfamily of amidotransferases that includes asparagine synthetase, glucosamine 6-phosphate synthase, and glutamate synthase, implying a common function in the four enzymes. Tyr74, on the other hand, is conserved only in glutamine PRPP amidotransferase sequences consistent with a specific role in interdomain coupling. The catalytic framework of key glutamine site residues supports the assignment of glutamine PRPP amidotransferase to a recently described Ntn (NH2-terminal nucleophile) hydrolase family of enzymes.

Adenosine Monophosphate↗
PubMed1995

Mechanism of corepressor-mediated specific DNA binding by the purine repressor.

The modulation of the affinity of DNA-binding proteins by small molecule effectors for cognate DNA sites is common to both prokaryotes and eukaryotes. However, the mechanisms by which effector binding to one domain affects DNA binding by a distal domain are poorly understood structurally. In initial studies to provide insight into the mechanism of effector-modulated DNA binding of the lactose repressor family, we determined the crystal structure of the purine repressor bound to a corepressor and purF operator. To extend our understanding, we have determined the structure of the corepressor-free corepressor-binding domain of the purine repressor at 2.2 A resolution. In the unliganded state, structural changes in the corepressor-binding pocket cause each subunit to rotate open by as much as 23 degrees, the consequences of which are the disengagement of the minor groove-binding hinge helices and repressor-DNA dissociation.

Allosteric Regulation↗
PubMed1995

Identification of the Bacillus subtilis pur operon repressor.

Transcription of the Bacillus subtilis pur operon is repressed in response to a signal of excess adenine. We have purified the repressor protein and have identified, cloned, and overexpressed the purR regulatory gene that controls transcription initiation of the operon. B. subtilis purR encodes a 62-kDa homodimer that binds to the pur operon control region. The PurR binding site which overlaps the promoter encompasses approximately 110 bp. The protein-DNA interaction is inhibited by 5-phosphoribosyl 1-pyrophosphate. A mutation that deletes the repressor binding site or one that disrupts purR abolishes binding activity in vitro and repression of transcription in vivo in response to the excess adenine signal. These results lead to a model in which an excess-adenine signal is transmitted to PurR via the 5-phosphoribosyl 1-pyrophosphate pool. In addition, purR is autoregulated. There is no structural or mechanistic similarity between the B. subtilis and Escherichia coli purine repressors.

Amino Acid Sequence↗
PubMed1995

A stable carbocyclic analog of 5-phosphoribosyl-1-pyrophosphate to probe the mechanism of catalysis and regulation of glutamine phosphoribosylpyrophosphate amidotransferase.

Glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase catalysis and regulation were studied using a new stable carbocyclic analog of PRPP, 1-alpha-pyrophosphoryl-2-alpha, 3-alpha-dihydroxy-4-beta-cyclopentane-methanol-5-phosphate (cPRPP). Although cPRPP competes with PRPP for binding to the catalytic C site of the Escherichia coli enzyme, two lines of evidence demonstrate that cPRPP, unlike PRPP, does not promote an active enzyme conformation. First, cPRPP was not able to "activate" Cys1 for reaction with glutamine or a glutamine affinity analog. The ring oxygen of PRPP may thus be necessary for the conformation change that activates Cys1 for catalysis. Second, binding of cPRPP to the C site blocks binding of AMP and GMP, nucleotide end product inhibitors, to this site. However, the binding of nucleotide to the allosteric site was essentially unaffected by cPRPP in the C site. Since it is expected that nucleotide inhibitors would bind with low affinity to the active enzyme conformation, the nucleotide binding data support the conclusion that cPRPP does not activate the enzyme.

Adenosine Monophosphate↗
PubMed1995

Analysis of the chicken GPAT/AIRC bidirectional promoter for de novo purine nucleotide synthesis.

GPAT and AIRC encode two enzymes that catalyze steps 1 and 6 plus 7, respectively, of the de novo purine biosynthetic pathway. The chicken genes are closely linked and divergently transcribed from an approximately 230-base pair intergenic region. The promoter was scanned by deletion mutagenesis in a bireporter vector that allowed assay of transcriptional activity in both directions in transfected HepG2 and chicken LMH cells. Three classes of deletions were obtained: those affecting bidirectional transcription, those predominantly affecting GPAT transcription, and those predominantly affecting AIRC transcription. Defects in bidirectional transcription resulted from removal of an initiator-like element overlapping the AIRC transcription start site, as well as deletions removing a series of GC and CCAAT boxes from the AIRC proximal half of the promoter and a CCAAT-containing segment from the GPAT side. Several regions in the GPAT proximal half of the promoter, including an octamer-like motif downstream from the transcription start site, were required predominantly for GPAT expression. Evidence for interaction of HeLa nuclear proteins with some of these sites was obtained by gel retardation, DNase I, and methylation interference assays. Overall, the results showed that the intergenic region is an integrated bidirectional promoter and that a novel initiator-like element plays a central role in coordinating expression of the divergently transcribed AIRC and GPAT genes.

Amidophosphoribosyltransferase↗