Biomedical subjects

H Zalkin

Publications and source records attributed to H Zalkin.

At least 37 records · Page 2Linked to original sources

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↗

PubMed1995

Formyltetrahydrofolate hydrolase, a regulatory enzyme that functions to balance pools of tetrahydrofolate and one-carbon tetrahydrofolate adducts in Escherichia coli.

The enzyme encoded by Escherichia coli purU has been overproduced, purified, and characterized. The enzyme catalyzes the hydrolysis of 10-formyltetrahydrofolate (formyl-FH4) to FH4 and formate. Formyl-FH4 hydrolase thus generates the formate that is used by purT-encoded 5'-phosphoribosylglycinamide transformylase for step three of de novo purine nucleotide synthesis. Formyl-FH4 hydrolase, a hexamer with 32-kDa subunits, is activated by methionine and inhibited by glycine. Heterotropic cooperativity is observed for activation by methionine in the presence of glycine and for inhibition by glycine in the presence of methionine. These results, along with previous mutant analyses, lead to the conclusion formyl-FH4 hydrolase is a regulatory enzyme whose main function is to balance the pools of FH4 and C1-FH4 in response to changing growth conditions. The enzyme uses methionine and glycine to sense the pools of C1-FH4 and FH4, respectively.

Allosteric Regulation↗

PubMed1994

Crystal structure of LacI member, PurR, bound to DNA: minor groove binding by alpha helices.

The three-dimensional structure of a ternary complex of the purine repressor, PurR, bound to both its corepressor, hypoxanthine, and the 16-base pair purF operator site has been solved at 2.7 A resolution by x-ray crystallography. The bipartite structure of PurR consists of an amino-terminal DNA-binding domain and a larger carboxyl-terminal corepressor binding and dimerization domain that is similar to that of the bacterial periplasmic binding proteins. The DNA-binding domain contains a helix-turn-helix motif that makes base-specific contacts in the major groove of the DNA. Base contacts are also made by residues of symmetry-related alpha helices, the "hinge" helices, which bind deeply in the minor groove. Critical to hinge helix-minor groove binding is the intercalation of the side chains of Leu54 and its symmetry-related mate, Leu54', into the central CpG-base pair step. These residues thereby act as "leucine levers" to pry open the minor groove and kink the purF operator by 45 degrees.

Amino Acid Sequence↗

PubMed1994

Mutagenesis of amino acid residues required for binding of corepressors to the purine repressor.

The corepressor-binding domain of the Escherichia coli purine repressor (PurR) is homologous with several periplasmic sugar-binding proteins. Four amino acids in PurR were investigated for a role in binding of corepressors. Three of the residues, Asp146, Arg196 and Asp275, are conserved in periplasmic binding proteins for ribose, glucose/galactose, and arabinose and function to bind sugars. A fourth amino acid, Trp147, required for corepressor binding to PurR, corresponds to residues in glucose/galactose, ribose, and arabinose that also have a role in sugar binding. The four mutations that were constructed perturbed the binding of both hypoxanthine and guanine thus providing evidence for a single corepressor site/PurR subunit. The decreased corepressor binding affinity resulted in reduced affinity of mutant repressors for operator DNA in vitro and decreased capacity for repression in vivo. The corepressor-binding site in PurR appears to be similar to the conserved ligand-binding sites in the three periplasmic sugar-binding proteins and in the LacI family of repressors.

Amino Acids↗

PubMed1994

Crystallization and preliminary X-ray analysis of an Escherichia coli purine repressor-hypoxanthine-DNA complex.

The purine repressor (PurR) is a DNA-binding protein, which together with a purine corepressor serves to regulate de novo purine and pyrimidine biosynthesis in Escherichia coli. PurR belongs to the structurally homologous lac repressor family of transcription regulators. A PurR-hypoxanthine-DNA complex has been crystallized, with DNA encompassing the high affinity purF operator site and which is 16 base-pairs long with 5'-deoxynucleoside overhangs on each complementary strand. The crystals diffract to better than 2.6 A and take the orthorhombic space group C222(1), with unit cell dimensions a = 175.9 A, b = 94.8 A and c = 81.8 A. The structure determination of this PurR-hypoxanthine-DNA complex will provide the first high resolution view of a Lacl member-DNA complex.

Bacterial Proteins↗

PubMed1994

Structure of the allosteric regulatory enzyme of purine biosynthesis.

Multi-wavelength anomalous diffraction (MAD) has been used to determine the structure of the regulatory enzyme of de novo synthesis of purine nucleotides, glutamine 5-phosphoribosyl-1-pyrophosphate (PRPP) amidotransferase, from Bacillus subtilis. This allosteric enzyme, a 200-kilodalton tetramer, is subject to end product regulation by purine nucleotides. The metalloenzyme from B. subtilis is a paradigm for the higher eukaryotic enzymes, which have been refractory to isolation in stable form. The two folding domains of the polypeptide are correlated with functional domains for glutamine binding and for transfer of ammonia to the substrate PRPP. Eight molecules of the feedback inhibitor adenosine monophosphate (AMP) are bound to the tetrameric enzyme in two types of binding sites: the PRPP catalytic site of each subunit and an unusual regulatory site that is immediately adjacent to each active site but is between subunits. An oxygen-sensitive [4Fe-4S] cluster in each subunit is proposed to regulate protein turnover in vivo and is distant from the catalytic site. Oxygen sensitivity of the cluster is diminished by AMP, which blocks a channel through the protein to the cluster. The structure is representative of both glutamine amidotransferases and phosphoribosyltransferases.

Adenosine Monophosphate↗

PubMed1994

Binding of purine nucleotides to two regulatory sites results in synergistic feedback inhibition of glutamine 5-phosphoribosylpyrophosphate amidotransferase.

Glutamine 5-phosphoribosylpyrophosphate amidotransferase from Escherichia coli is subject to synergistic feedback regulation by adenine and guanine nucleotides. Inhibition assays and equilibrium binding measurements have established that synergistic inhibition by AMP and GMP results from synergistic binding to two sites/enzyme subunit in the homotetramer. Although each nucleotide can bind to both sites, analyses of the wild type and mutant enzymes indicate that binding of GMP to an A (allosteric) site and AMP to a proximal C (catalytic) site are necessary for synergistic inhibition. K326Q and P410W amino acid replacements result in decreased binding affinity for GMP and AMP and lead to corresponding reductions in feedback inhibition. The K326Q A site mutation results not only in decreased affinity of GMP for the mutant A site but also has an adverse effect on AMP affinity for the C site. Similarly, the P410W C site mutation has a detrimental effect on binding of AMP to the mutant C site and also on affinity of GMP to the A site. The fact that a mutation in one site affects binding of nucleotides to both sites provides further evidence for synergistic binding of nucleotides.

Adenosine Monophosphate↗

PubMed1994

Two genes for de novo purine nucleotide synthesis on human chromosome 4 are closely linked and divergently transcribed.

A cDNA encoding human glutamine phosphoribosylpyrophosphate amidotransferase for step one in de novo purine nucleotide synthesis was cloned, sequenced, and expressed in Chinese hamster ovary cells to yield functional enzyme. Enzyme function was dependent upon removal of an 11-amino-acid propeptide. A mutant enzyme having three propeptide amino acid replacements was not processed and was not active. The human genes GPAT, encoding the amidotransferase, and AIRC, encoding a bifunctional enzyme for steps six and seven in the pathway, were cloned and characterized. GPAT and AIRC are closely linked and divergently transcribed from an intergenic region of approximately 625 base pairs. Expression of a luciferase reporter from the GPAT promoter was approximately 3-4-fold higher than from the AIRC promoter. The GPAT gene was mapped to the q12 region of chromosome 4.

Amidophosphoribosyltransferase↗

PubMed1994

Regulation of Escherichia coli purA by purine repressor, one component of a dual control mechanism.

Escherichia coli purA encodes adenylosuccinate synthetase, one of two enzymes required for synthesis of AMP from IMP. purA is subject to two- to threefold regulation by purR and about twofold regulation by a purR-independent mechanism. The 5'-flanking region of purA confers purR-dependent transcriptional regulation of purA but not the purR-independent regulation. Two operator sites in the 5'-flanking region which bind purine repressor in vitro and are required for in vivo regulation were identified. The purR-independent regulation may be posttranscriptional. It is now established that all transcription units involved in de novo synthesis of purine nucleotides, nine pur operons, as well as purR itself and guaBA, are subject to purR control.

Adenosine Monophosphate↗

PubMed1994

Role of the purine repressor hinge sequence in repressor function.

A protease-hypersensitive hinge sequence in Escherichia coli purine repressor (PurR) connects an N-terminal DNA-binding domain with a contiguous corepressor-binding domain. Binding of one molecule of dimeric repressor to operator DNA protects the hinge against proteolytic cleavage. Mutations in the hinge region impair repressor function in vivo. Several nonfunctional hinge mutants were defective in low-affinity binding to operator DNA in the absence of corepressor as well as in high-affinity corepressor-dependent binding to operator DNA, although binding of corepressor was similar to binding of the wild-type repressor. These results establish a role for the hinge region in operator binding and lead to a proposal for two routes to form the holoPurR-operator complex.

Amino Acid Sequence↗

PubMed1993

Expression, purification, and kinetic characterization of recombinant human adenylosuccinate lyase.

Adenylosuccinate adenosine 5'-monophosphate lyase (EC 4.3.2.2; ASL) catalyzes two distinct reactions in adenosine 5'-monophosphate (AMP) biosynthesis. A S413P mutation in ASL segregates with mental retardation in an affected family (Stone, R. L., Aimi, J., Barshop, B. A., Jaeken, J., Van den Berghe, G., Zalkin, H., and Dixon, J. E. (1992) Nature Genet. 1, 59-63). ASL and S413P ASL have been expressed, purified, and kinetically characterized. Lowering the Escherichia coli growth temperature to 25 degrees C and the concentration of inducer, isopropyl-1-thio-beta,D-galactopyranoside, to 40 microM was necessary for synthesis of soluble, tetrameric enzymes. The recombinant enzymes were purified to homogeneity using anion exchange chromatography followed by chromatography on Blue 2A Sepharose. At pH 7.0 and 25 degrees C, the kcat for cleavage of 5-amino-4-imidazole-N-succinocarboxamide ribotide (SAI-CAR) by ASL was 90 s-1 with a Km of 2.35 microM. The kcat for adenylosuccinate (SAMP) cleavage was 97 s-1 with a Km of 1.79 microM. The catalytic mechanism involved one general base catalyst (pK alpha = 6.4) and one general acid catalyst (pK alpha = 7.5). ASL follows an ordered uni-bi reaction mechanism with fumarate released first. 5-Amino-4-imidazolecarboxamide ribotide (AICAR) and AMP were competitive with SAICAR and SAMP (Ki[AICAR] = 11.3 microM; Ki[AMP] = 9.2 microM), whereas fumarate inhibited noncompetitively (Kii = 2.3 mM, Kis = 2.8 mM). The competitive inhibition by AICAR and AMP suggests a single active site that binds both SAICAR and SAMP. The kinetic constants at pH 7.0, 25 degrees C and the kcat/Km versus pH profiles for ASL and S413P ASL were very similar. These results are consistent with S413P being a structural rather than a catalytic defect.

Adenylosuccinate Lyase↗

PubMed1993

Identification of sites for feedback regulation of glutamine 5-phosphoribosylpyrophosphate amidotransferase by nucleotides and relationship to residues important for catalysis.

Glutamine phosphoribosylpyrophosphate amidotransferase, the key regulatory enzyme for de novo purine nucleotide synthesis, is subject to feedback regulation by adenine and guanine nucleotides. Affinity labeling with 5'-p-fluorosulfonylbenzoyladenosine (FSBA) and 8-azidoadenosine 5'-monophosphate (N3-AMP) was used to identify purine nucleotide sites for feedback control of the Escherichia coli amidotransferase. FSBA inactivated the amidotransferase with saturation kinetics. Specificity for inactivation was shown by the covalent attachment of 2.0-2.4 eq of [3H] sulfobenzoyladenosine (SBA) per subunit and protection by GMP and AMP against inactivation and incorporation of [3H]SBA. Six chymotryptic peptides modified with [3H]SBA were isolated and identified by differential labeling followed by high performance liquid chromatography and radioactivity. Mass spectrometry and Edman degradation analysis were used to identify 5 residues that were covalently modified by [3H]SBA: Tyr74, Tyr258, Lys326, Tyr329, and Tyr465. Tyr258 was also modified by N3-AMP. Mutant enzymes K326Q and Y329A had activity similar to that of the wild type enzyme. However, both mutants exhibited decreased sensitivity to inhibition by GMP and decreased binding of GMP but were inhibited by AMP. Mutant enzymes Y74A and Y258F were normally feedback-inhibited but were defective in glutamine amide transfer and synthase functions, respectively. Therefore Tyr74 and Tyr258 are important for activity and modification by FSBA and N3-AMP accounts for enzyme inactivation. These results localize residues important for catalysis in close proximity to a site for nucleotide binding. Two additional mutant enzymes, G331I and N351A, were constructed which were refractory to inhibition by GMP with little change in inhibition by AMP. A replacement of Tyr465 indicates that this residue is not essential for catalysis or feedback inhibition. Overall, these results are interpreted in terms of a two-nucleotide site model with Lys326, Tyr329, Gly331, and Asn351 defining a site required for inhibition by GMP. A second nucleotide site not affinity labeled by analogs is very close to or overlaps with the catalytic site.

Adenosine↗