Progress 10/01/99 to 09/30/04
Outputs
During the previous 34 years the PI has carried out basic studies to understand gene expression and enzyme function. Specific interests have included (I) genes and enzymes in the biosynthesis of aromatic amino acids, phenylalaine, trosine and tryptophan; (ii) genes and enzymes involved in synthesis of purine nucleotides; (iii) structure and function of glutamine amidotransferase enzymes. Genes required for synthesis of purines were identified and isolated for the first time from microorganisms, chickens and humans. The molecules that control expression of genes for purine synthesis were identified for the first time in two bacteria, E. coli and B. subtilis. Crystal structures of these regulatory proteins were determined and the biochemical mechanisms for interaction with DNA control sites determined. This information has provided a rather detailed picture of how structure dictates function, that is how expression of genes for purine biosynthesis is controlled to meet
cellular needs. Research on glutamine amidotransferases has led to a detailed understanding of how this important enzyme family uses the nitrogen atom in the amino acid glutamine for the biosynthesis of essentially all nitrogen containing biomolecules in the cell.
Impacts
The PI's research is part of the knowledge base used for drug discovery and food flavor production. One glutamine amidotransferase is a current drug target for a large pharmaceutical company. The production of a major seasoning ingredient for Asian foods is based on the PI's research on purine biosynthesis in Bacillus subtilis.
Publications
- Li, S., Smith, J.L., and Zalkin, H. 1999. Mutational analysis of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase propeptide processing. J. Bacteriol. 181, 1403-1408.
- Chen, S. Burgner, J.W., Krahn, J.M., Smith, J.L., and Zalkin, H., 1999. Trptophan florescence monitors multiple conformational changes required for glutamine phosphoribosylpyrophosphate amidotransferase interdomain signaling and catalysis. Biochemistry 38, 11659-11669.
- Bera, A.K., Chen, S., Smith, J.L., and Zalkin, H., 1999. Interdomain signaling in glutamine phosphoribosylpyrophosphate amidotransferase. J. Biol. Chem. in press.
- Sinha, S., Rappu, P., Lange, S.C., Mantsala, P., Zalkin, H., and Smith, J.L., 1999. Crystal structure of Abcillus subtilis YabJ, a purine regulatory protein and member of the highly conserved YjgF Family. Proc. Natl. Acad. Sci USA, in press.
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Progress 10/01/98 to 09/30/99
Outputs
During the previous 34 years the PI has carried out basic studies to understand gene expression and enzyme function. Specific interests have included (i) genes and enzymes in the biosynthesis of the aromatic amino acids, phenylalaine, tyrosine and tryptophan; (ii) genes and enzymes involved in synthesis of purine nucleotides; (iii) structure and function of glutamine amidotransferase enzymes. Genes required for synthesis of purines were identified and isolated for the first time from microorganisms, chickens and humans. The molecules that control expression of genes for purine synthesis were identified for the first time in two bacteria, E. coli and B. subtilis. Crystal structures of these regulatory proteins were determined and the biochemical mechanisms for interaction with DNA control sites determined. This information has provided a rather detailed picture of how structure dictates function, that is how expression of genes for purine biosynthesis is controlled to
meet cellular needs. Research on glutamine amidotransferases has led to a detailed understanding of how this important enzyme family uses the nitrogen atom in the amino acid glutamine for the biosynthesis of essentially all nitrogen containing biomolecules in the cell.
Impacts
The PI's research is part of the knowledge base used for drug discovery and food flavor production. One glutamine amidotransferase is a current drug target for a large pharmaceutical company. The production of a major seasoning ingredient for Asian foods is based on the PI's research on purine biosynthesis in Bacillus subtilis.
Publications
- Sinha, S., Rappu, P., Lange, S.C., Mantsala, P., Zalkin, H., and Smith, J.L. 1999. Crystal structure of Bacillus subtilis YabJ, a purine regulatory protein and member of the highly conserved YjgF family. Proc. Natl. Acad. Sci. USA 96, 13074-13079.
- Li, S., Smith, J.L., and Zalkin, H. 1999. Mutational analysis of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase propeptide processing. J. Bacteriol. 181, 1403-1408.
- Chen, S., Burgner, J.W., Krahn, J.M., Smith, J.L., and Zalkin, H., 1999. Tryptophan fluorescence monitors multiple conformational changes required for glutamine phosphoribosylpyrophosphate amidotransferase interdomain signaling and catalysis. Biochemistry 38, 11659-11669.
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Progress 10/01/97 to 09/30/98
Outputs
The objectives of this research are to characterize the regulatory molecules that control the production of purine nucleotides. We investigate the two key regulatory molecules that control purine nucleotide synthesis in E. coli and other organisms. The purine repressor (PurR) regulates expression of all of the genes in the biosynthetic pathway leading to the synthesis of adenine and guanine nucleotides. PurR is a member of the Lac family of transcriptional regulators. Glutamine PRPP amidotransferase (GPAT) is the key regulatory enzyme in the biosynthetic pathway. GPAT is one of about 20 glutamine amidotransferases. We use x-ray structural information to guide biochemical analysis of function. We have recently investigated how binding of purine corepressor to a site 40 angstrom from the DNA binding site increases PurR binding affinity to DNA control sites. The data indicate that PurR exists in an equilibrium between "open" and "closed" conformations having low affinity
and high affinity, respectively, for DNA binding. Corepressor was found to shift the allosteric equilibrium in favor of the closed conformation. With respect to GPAT, studies are currently in progress to examine conformational transitions that are important for catalysis. The basis for this work is the recent determination of x-ray structures of a ligand-free inactive conformer and an enzyme-substrate complex that reflects the fully active conformation. Several structural rearrangements distinguish the two conformers. We are using intrinsic tryptophan fluorescence to examine intermediate steps in the transition between inactive and active conformers. We expect these studies on PurR and GPAT to provide basic information that will advance our understanding of the Lac family of transcriptional regulators and the glutamine amidotransferase enzyme family, respectively.
Impacts
(N/A)
Publications
- Arvidson, D. N., Lu, F., Faber, C., Zalkin, H., and Brennan, R. G. 1998. The structure of PurR mutant L54M shows an alternative route to DNA kinking. Nature Structural Biology 5, 436-441.
- Lu, F., Schumacher, M. A., Arvidson, D. N., Haldimann, A., Wanner, B. L., Zalkin, H., and Brennan, R. G. 1998. Structure-based redesign of corepressor specificity of the Escherichia coli purine repressor by substitution of residue 190. Biochemistry 37, 971-982.
- Shin, B. S., Stein, A., and Zalkin, H. 1997. Interaction of Bacillus subtilis purine repressor with DNA. J. Bacteriol. 179, 7394-7402.
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Progress 10/01/96 to 09/30/97
Outputs
The objectives of this research are to determine how the synthesis of purine nucleotides is controlled. We are investigating the two key regulatory molecules that control purine nucleotide synthesis in E. coli and other organisms. The purine repressor (PurR) regulates expression of all the genes in the biosynthetic pathway leading to synthesis of adenine and guanine nucleotides, and glutamine PRPP amidotransferase (GPAT) is the regulatory enzyme in the biosynthetic pathway. We use x-ray structural information to guide biochemical analysis of function. We want to determine how groups at the 2- and 6-positions of the purine ring establish the binding specificity of purines to PurR. We have determined the structural basis for guanine and hypoxanthine corepressor specificity at the 2-position. The higher binding affinity of guanine to PurR compared to hypoxanthine and the ability of PurR to discriminate against 2-oxopurines (xanthine) results from a water mediated contact
with the exocyclic N-2 of guanine and the better electrostatic complementarity of the guanine base with the corepressor-binding pocket. Next, we will determine how the 2-position of the purine ring influences binding specificity. We have reported two major advances in GPAT catalysis and regulation. GPAT is a member of a glutamine amidotransferase enzyme family in which the amide N of glutamine is used to incorporate N into precursor molecules used for biosynthesis. There has been a long standing question how the N is moved from glutamine to the acceptor substrate. We determined that binding of PRPP promotes a dramatic enzyme structural change resulting in a tunnel that connects active sites for the two substrates, glutamine and PRPP. This indicates that glutamine is hydrolyzed and the resulting ammonia is channeled through a 16 angstrom tunnel for reaction with PRPP. Accompanying formation of the tunnel, the PRPP site is closed to exclude water which would hydrolyze PRPP. The second
major development concerns the detailed mechanism for feedback regulation of GPAT by nucleotides. We identified two distinct nucleotide sites for each GPAT subunit and determined the basis for nucleotide binding specificity. Binding of ADP to an A-site and GMP to a C-site results in a specific nucleotide-nucleotide interaction that increases binding affinity by more than 20-fold and inhibition by more than 100-fold compared to the individual nucleotides. This synergistic inhibition has very specific nucleotide specificity which is now explained by a new x-ray structural analysis.
Impacts
(N/A)
Publications
- Kim, J.H., Krahn, J.M., Smith, J.L., and Zalkin, H. 1996 Structure and Function of the Glutamine Phosphoribosylpyrophosphate Amidotransferase Glutamine Site and Communication with the Phosphoribosylpyrophosphate Site. J. Biol. Chem. 271, 15549-15557.
- Glasfeld, A., Schumacher, M.A., Choi, K.-Y., Zalkin, H. and Brennan, R.G. 1997 A Positively Charged Residue Bound in the Minor Groove Does Not Alter the Bending of a DNA Duplex. J. Am. Chem. Soc. 118, 13073-13074.
- Chen, S., Nagy, P.L. and Zalkin, H. 1997. Role of NRF-1 in Bidirectional Transcription of the Human GPAT-AIRC Purine Biosynthesis Locus. Nucleic Acids Res. 25, 1809-1816.
- Zalkin, H. 1997 Formyltetrahydrofolate Hydrolase from Escherichia coli. Methods in Enzymol.: Vitamins and Coenzymes, Part K 281, 214-218.
- Krahn, J.M., Kim, J.H., Burns, M.R., Parry, R.J., Zalkin, H., and Smith, J.L. 1997 Coupled Formation of an Amidotransferase Ammonia Channel and a Phosphoribosyltransferase Active Site. Biochemistry 36, 11061-11068.
- Chen, S., Tomchick, D.R., Wolle, D., Hu, P., Smith, J.L., Switzer, R.L., and Zalkin, H. 1997 Mechanism of the Synergistic Endproduct Regulation of Bacillus subtilis Glutamine Phosphoribosylpyrophosphate Amidotransferase by Nucleotides. Biochemistry 36, 10718-10726.
- Schumacher, M.A., Glasfeld, A., Zalkin, H., and Brennan, R.G. 1997 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. J. Biol. Chem. 272, 22648-22653.
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Progress 10/01/95 to 09/30/96
Outputs
Glutamine PRPP amidotransferase catalyzes the first reaction in the purine biosynthetic pathway and is the key regulatory enzyme in the pathway. The enzyme has two domains that function in catalysis, an N-domain that hydrolyzes glutamine (glutamine # glutamate + NH3) and a C-domain that catalyzes the reaction NH3 + P-Rib-PP # P-Rib-NH2 + PPi. The coupling of these two half reactions results in the overall reaction, glutamine + P-Rib-PP # glutamate + P-Rib-NH2 + PPi. The two half reactions are tightly coupled, that is, glutamine hydrolysis is dependent upon the binding of P-Rib-PP to the C-domain. Two key questions are how coupling takes place and how the NH3 released from glutamine can be sequestered so that it doesn#t dissociate from the enzyme or exchange with external NH3. We have determined the x-ray structure of the glutamine site in the N-domain and have an initial picture of how coupling is achieved. In the absence of P-Rib-PP the enzyme is in a basal state
having a low affinity for glutamine and a low glutaminase activity. Binding of P-Rib-PP results in an active conformation with a functional glutamine site. Amino acids involved in the binding and reaction of glutamine were identified in the structure. Thus, binding of P-Rib-PP to the C-domain triggers a structural change that makes the glutaminase half reaction possible. We are now determining how the NH3 released from glutamine is sequestered so that it must react with P-Rib-PP.
Impacts
(N/A)
Publications
- Kim, J. H., Krahn, J. M., Tomchick, D. R., Smith, J. L, and Zalkin, H. 1996. Structure and function of the glutamine phosphoribosylpyrophosphate amidotransferase glutamine site and communication with the phosphoribosylpyrophosphate site. J.
- Gavalas, A. And Zalkin, H. 1995 Analysis of the chicken GPAT/AIRC bidirectional promoter for de novo purine nucleotide synthesis. J. Biol. Chem. 2403-2410.
- Nagy, P.L., Marolewski, A., Benkovic, S.J., and Zalkin, H. 1995. Formyltetrahydrofolate hydrolase, a regulatory enzyme that functions to balance pools of tetrahydrofolate and one carbon tetrahydrofolate adducts in Escherichiacoli. J. Bacte
- Zalkin, H., and Nygaard, P. 1995. Biosynthesis of purine nucleotides. IN Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, Second Ed..(
- F.C. Neidhardt, R. Curtiss, C.A. Gross, J.L. Ingraham, E.C.C. Lin, E.B. Low, Kim, J.H., Wolle, D., Haridas, K., Parry, R.J., Smith, J., and Zalkin, H. 1995 A stable carboxyclic analog of 5-phosphoribosyl-1-pyrophosphate to probe the mechanism of catalysis and regulation of glutamine phosphoribosylpyrophosphate amido
- Weng, M., Nagy, P.L., and Zalkin, H. 1995. Identification of the Bacillus subtilis pur operon repressor. Proc. Natl. Sci. (USA) 92:7455-7459.
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Progress 10/01/94 to 09/30/95
Outputs
We have continued studies to elucidate the regulatory mechanisms that control purine nucleotide synthesis in bacterial and animal cells. There have been several important developments. First, there is new information about how the E. coli purine repressor (PurR) binds to DNA to regulate gene expression. A cellular purine molecule, a corepressor, initially binds to the dimeric repressor protein and increases its DNA binding affinity about 15-fold. The x-ray structure of the corepressor-free PurR has now been compared with the structure of the PurR#corepressor#DNA complex. The results indicate that subunit rotations of about 23# accompany binding of the corepressor and are responsible for increased DNA binding affinity. This is the first example for how a small sensor molecule, in this case a purine corepressor, interacts with a regulatory protein and transmits a signal to a distal DNA binding domain that changes protein-DNA binding affinity. A second important
development was the identification, cloning and overproduction of a repressor protein that controls purine biosynthesis in Bacillus subtilis. There is no structural or mechanistic similarity between B. Subtilis and E. coli purine repressors. The small molecular coeffector for B. subtilis PurR is 5-phosphoribosyl-1-pyrophosphate (PRPP). A model was worked out to explain how the pool of cellular purines modulates PurR-DNA binding and gene regulation via PRPP. Other work has provided new information on the major regulatory enzyme of the pathw.
Impacts
(N/A)
Publications
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Progress 10/01/93 to 09/30/94
Outputs
This research has focused on the regulatory mechanisms that control purine nucleotide synthesis in bacterial and animal cells. In bacteria gene regulation and enzyme regulation control the production of purine nucleotides. We have contributed to the characterization of the two key regulatory proteins that control gene expression and enzyme activity for purine synthesis in E. coli. As a result we have for the first time an initial understanding at the molecular level of how purine production is regulated in a cell. Gene expression is modulated by a repressor protein, PurR, and glutamine PRPP amidotransferase is the important regulatory enzyme in the pathway. We have developed procedures for the overproduction and purification of sufficient amounts of PurR for structural characterization. In the first of two important developments, the x-ray structure of a PurR.hypoxanthine.operator DNA ternary complex has very recently been solved. This structure provides initial
information about how the corepressor hypoxanthine, binds to the protein and how the repressor binds to DNA. Seven amino acids are implicated in binding hypoxanthine. Site-directed mutagenesis has confirmed the role of two of these amino acids, Asp(superscript 275) and Arg(superscript 196) in corepressor binding. The x-ray structure provides important information about repressor-DNA interaction.
Impacts
(N/A)
Publications
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Progress 10/01/92 to 09/30/93
Outputs
There are 14 steps in the pathway for de novo synthesis of purine nucleotides. Although the pathway is identical in all organisms that synthesize purine nucleotides de novo, there are different patterns of gene organization and different mechanisms for gene and enzyme regulation in bacteria, plants and animals. Our first understanding of how gene and enzyme regulation modulate the production of purines is emerging in one organism, Escherichia coli. This laboratory has made significant contributions to the understanding of purine gene and enzyme regulation in E. coli and to further expand this knowledge to other organisms. There are two important regulatory elements for purine gene regulation in E. coli, a repressor protein (PurR) and a DNA site (an operator) to which the repressor binds in order to modulate transcription. The PurR-operator interaction regulates the expression of all 14 steps in the de novo pathway. We have developed a procedure to overexpress the
repressor and purify the protein in amounts sufficient for structural characterization. The repressor was found to have two well defined domains, a 50 amino acid N-terminal DNA binding domain connected by a short hinge to a 290 amino acid domain with functions for corepressor binding and dimerization. We have separated the two domains for functional analysis and for crystallization and structure determination.
Impacts
(N/A)
Publications
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Progress 10/01/91 to 09/30/92
Outputs
There are 14 steps for de novo synthesis of purine nucleotides. The pathway appears identical in all organisms that synthesize purine nucleotides, but different patterns of gene organization, and different mechanisms for gene regulation and enzyme regulation are found in bacteria, plants and animals. We have previously characterized these patterns. One objective is to obtain a complete understanding of purine nucleotide gene regulation in E.coli. We now know that two control elements (a repressor and an operator) regulate 8 of 9 transcription units (operons) containing the genes for purine nucleotide synthesis in E.coli. The repressor is encoded by purR. The purified repressor binds to a 16 bp conserved operator site in the 8 operons and regulates transcription. In 7 of the 8 operons, binding of repressor to an operator site in the promoter region regulates transcription initiation. In one gene, purB, the operator is located 242 bp downstream from the promoter and
regulates transcription by a roadblock mechanism, that is by blocking transcription elongation. The roadblock is less efficient than control of transcription initiation. The purB gene functions at a position in the pathway that is required not only for de novo synthesis but also for synthesis by so-called "salvage" reactions. We believe that the roadblock mechanism is well adapted to provide modest regulation where it is important to maintain basal level expression even when most enzymes of the de novo pathway are shut down.
Impacts
(N/A)
Publications
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Progress 10/01/90 to 09/30/91
Outputs
In all organisms there are 14 steps for the de novo synthesis of purine nucleotides. Although the pathway is invariant genetic organization and regulation of expression vary. We have investigated the mechanism for regulation of the genes for the initial 10 steps of the pathway to IMP which is the first purine nucleotide intermediate. In E. coli these genes are organized in 7 unlinked operons subject to regulation by gene purR. We have determined that purR encodes a repressor protein that binds to a conserved 16 bp operator site in each of the 7 operons. Thus, binding of repressor protein to each operator negatively coregulates expression. Gene purR which encodes the repressor protein is itself autoregulated. The mechanism of autoregulation requires two operator sites, purR(subscript 01) and purR(subscript O2), each located in an unusual position downstream of the promoter in the mRNA. We do not understand the significance of the dual operators and their position in
the gene. In other work a system was developed for overproduction of the repressor and purification to homogeneity. This allowed identification of two purine corepressors, hypoxanthine and guanine which are required to promote binding of the repressor to operator DNA. Hypoxanthine and guanine thus signal the purine status of the cell to the repressor-operator regulatory system which sets the level of gene expression for synthesis of purine nucleotides.
Impacts
(N/A)
Publications
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Progress 10/01/89 to 09/30/90
Outputs
Strategies were developed to clone cDNAs encoding the enzymes required for the 14 reactions of de novo purine nucleotide synthesis from animals. Using functional complementation of E. coli pur mutants with an expression library and PCR cloning, we have isolated cDNAs encoding enzymes for 11 of the 14 steps in the pathway to AMP and GMP. A cDNA library from chicken liver was employed since birds use the de novo purine nucleotide pathway not only for biosynthesis but also for excretion of excess nitrogen as uric acid and thus contain elevated enzyme levels. The complete sequences of the following enzymes were determined for the first time from an animal: glutamine PRPP amidotransferase (reaction 1), GAR synthetase-AIR synthetase-GAR transformylase (reactions 2,5,3), SAICAR synthetase-AIR carboxylase (reactions 7,6), adenylosuccinate lyase (reactions 8,12) and AICAR transformylase-IMP cyclohydrolase (reactions 9,10). Avian glutamine PRPP admidotransferase is 40%
identical to the sequence of the Bacillus subtilis enzyme. It appears likely that the avian enzyme requires two posttranslational modifications to acquire activity: removal of an 11 amino acid pro-peptide and assembly of an Fe-S cluster. The role of these modifications is under investigation. The relationships of enzyme defects to human diseases are also under investigation.
Impacts
(N/A)
Publications
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Progress 10/01/88 to 09/30/89
Outputs
Research from this laboratory is concerned with the structure, function and regulation of glutamine amidotransferase genes and enzymes. In this project period we have studied the regulation of an operon encoding 10 enzymes for purine nucleotide synthesis from Bacillus subtilis; cloned and characterized an amidotransferase, asparagine synthetase, from chinese hamster; studied the catalytic mechanism for the amidotransferase, amidophosphoribosyltransferase, that catalyzes the first reaction for purine nucleotide synthesis in E. coli; and established the role of a [4Fe-4S] cluster in B. subtilis amidophosphoribosyltransferase. E. coli and B. subtilis amidophosphoribosyltransferase are homologous enzymes that differ in two substantial respects: the B. subtilis enzyme is synthesized with an 11 amino acid pro-peptide and it contains a non-catalytic [4Fe-4S] cluster. We have now demonstrated in collaboration with Dr. R. L. Switzer's laboratory, University of Illinois, that
the Fe-S cluster is a site for oxidative inactivation of the enzyme. Oxidative inactivation is the initiating, rate limiting step for enzyme turnover. Thus, when B. subtilis cells are starved for a nutrient and initiate the developmental pathway leading to sporulation, biosynthesis of purine nucleotides is no longer needed. Biosynthetic capacity is turned off by oxidative inactivation and degradation of the first enzyme in the pathway, amidophosphoribosyltransferase.
Impacts
(N/A)
Publications
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Progress 10/01/87 to 09/30/88
Outputs
Research from this laboratory is concerned with the structure, function and regulation of glutamine amidotransferase genes and enzymes. We have previously cloned and sequenced the entire set of 12 genes for de novo purine nucleotide synthesis in Bacillus subtilis. This is the first time that all of the genes for purine biosynthesis have been isolated from any organism. We have now initiated a study of the regulation of de novo purine nucleotide synthesis in B. subtilis. The 12 gene B. subtilis pur operon is regulated independently by adenine and guanine nucleotides. Adenine nucleotides repress transcription initiation and guanine nucleotides regulate transcription by a termination-antitermination mechanism in a 242 nucleotide untranslated leader region preceding the first structural gene. We have identified the intact, prematurely terminated leader mRNA transcript of 200 nucleotides in length in guanine-grown cells. Adenine nucleotides repressed the formation of this
mRNA. This provides further evidence for the independent regulation of pur operon transcription by adenine and guanine nucleotides. The accumulated mRNA had a half life of about 0.7 min and was degraded in a series of discrete steps, resulting in the accumulation of stable intermediates in vivo. The degradation pathway for this mRNA was determined. Degradation was initiated by a series of endonucleolytic cleavages followed by 3' to 5' exonuclease catalyzed degradation.
Impacts
(N/A)
Publications
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Progress 10/01/86 to 09/30/87
Outputs
Research from this laboratory is concerned with the structure, function and regulation of glutamine amidotransferase genes and enzymes. There are 3 glutamine amidotransferases in the de novo pathway for purine nucleotide synthesis. We have, for the first time, cloned and sequenced an entire set of genes from a single organism for the synthesis of IMP. The nucleotide sequence of a more than 13 kb region of the Bacillus subtilis chromosome was determined and found to contain a cluster of 12 genes encoding enzymes that catalyze the 10 reactions for de novo synthesis of IMP from P-Rib-PP. The cluster is likely an operon that is organized into 3 groups of overlapping genes followed by the last gene: purEKB-purC(orf) QLF-purMNH(J)-purD. RNA analyses indicate that synthesis of purine nucleotides is regulated independently by adenine and guanine nucleotides. Adenine nucleotides regulate transcription initiation. Guanine nucleotides regulate transcription by a
termination-antitermination mechanism in a 242-nucleotide 5' untranslated mRNA leader region. Groups of overlapping genes, regulated at least in part by transcription termination-antitermination is likely to be a common theme for genetic organization and regulationof biosynthetic genes in Bacilli. Sequence comparisons provide evidence for homology of monofunctional purine nucleotide biosynthetic enzymes from B. subtilis with the corresponding multifunctional enzymes from yeast and Drosophila.
Impacts
(N/A)
Publications
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Progress 10/01/85 to 09/30/86
Outputs
Glutamine amidotransferases are a family of enzymes involved in the biosynthesisof amino acids, purine and pyrimidine nucleotides, amino sugars and coenzymes. These enzymes provide a main route for incorporation of nitrogen into these molecules. We have reported studies on the structure and function of three amidotransferases: anthranilate synthase, CTP synthetase and amidophosphoribo-syltransferase. The mechanism for activation of glutamine involves formation of a covalent glutaminyl enzyme intermediate with an active site cysteine. We have inferred the essential function of a single conserved histidine residue in the glutamine amide transfer domain of anthranilate synthase. Replacement of histidine 170 by tyrosine reduced the capacity to form the covalent glutaminyl intermediate. Other experiments suggested that histidine 170 functions as a general base to facilitate formation of the cysteine nucleophile (B: + cys-SH == BH + cys-S). During the past year we
reported the cloning and sequence determination of pyrG which encodes CTP synthetase. From the derived amino acid sequence we identified the CTP synthetase glutamine amide transfer (GAT) domain. The CTP synthetase GAT domain is homologous to the GAT domains in five other amidotransferases. Amidophosphoribosyltransferase from B. subtilis has a 4Fe-4S cluster of unknown function.
Impacts
(N/A)
Publications
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Progress 01/01/85 to 09/30/85
Outputs
We reported results of continuing studies on (i) gene regulation and (ii) the structure/function of the glutamine amide transfer (GAT) function of glutamine amidotransferase enzymes. With regard to gene regulation, we analyzed the control region of yeast gene TRP5, involved in biosynthesis of tryptophan, and a bacteria gene purF, involved in de novo purine nucleotide synthesis. Yeast TRP5 was found to contain a promoter region extending from nucleotide -188 to -29 (transcription start at +1). The promoter contains two elements, an upstream activation sequence (UAS), nucleotides -188 to -114 and a TATA sequence at -40. Regulatory regions essential for "general control" overlap with the promoter. E. coli purF exhibits a complex promoter that requires DNA between nucleotides -96 and -71 in addition to the Pribnow box at -10. A control site, having dyad symmetry, overlaps the Pribnow box and presumably serves to allow binding of an as yet uncharacterized repressor
protein. Regulation of purF expression is at the level of transcription. In addition purF is regulated by the availability of amino acids by "stringent control". Major new insights were developed for understanding the GAT function of the amidotransferase enzyme family. A homologous GAT domain has now been recognized and detected in four different enzymes: GMP synthetase, carbamoyl-P synthetase, anthranilate synthase and p-aminobenzoate synthase. Ideas were developed to explain the pattern of GAT fusions in these enzymes.
Impacts
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Publications
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Progress 01/01/84 to 12/30/84
Outputs
Saccharomyces cervisiae gene TRP2 and TRP3 encoding bifunctional anthranilate synthase-indol-3-glycerol phosphate synthase (AS-InGPS) and ADE4 encoding glutamine phosphoribosylpyrophosphate amidotransferase (APRTase) were cloned and sequenced. The cloning and sequencing provide the framework for studies on gene regulation and analysis of protein structure-function. TRP2 and TRP3 are subject to regulation by the general amino acid control system. Flanking sequences possibly involved in general control were identified. TRP3-encoded AS subunit II and InGPS are homologus with the corresponding E. coli trpG and trpC proteins. The primary structure of the bifunctional TRP3 protein has the arrangement AS subunit II/11 amino acid connector/InGPS. ADE4-encoded APRTase cysteine involved in glutamine amide transfer and concluded that the yeast enzyme lacks a sequence that functions as a 4Fe-4S binding site. Therefore, FE-S is not obligatory to the function of APRTase from
eukaryotes. Active site cysteine-12 in B. subtilis APRTase was replaced by Phe using site-directed mutagenesis. This replacement abolished glutamine-dependent activity and prevented the normal NH(2)-terminal undecapeptide processing. NH(3)-dependent APRTase functioned for in vivo purine nucleotide synthesis when the NH(3) level was high but not when the NH(3) concentration in the media was less than 5 mM.
Impacts
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Publications
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Progress 01/01/83 to 12/30/83
Outputs
Studies on the structure, function and regulation of genes encoding glutamine amidotransferase enzymes were continued and a new project of yeast tryptophan genes/enzymes was initiated. The structure and function of Escherichia coli glutamate synthase were studied by ligand binding and chemical modification experiments. Binding sites for the three substrates, NADP, glutamine, and 2-oxoglutarate were characterized. Residues essential for the binding of NADP and glutamine were identified by chemical modification. A main conclusion was that 1 eq of NADP and 0.5 eq of 2-oxoglutarate bind and function in the NH(3)-dependent synthesis of glutamate. For the glutamine-dependent sythesis of glutamate, glutamine promotes a conformation change which increases the binding of 2-oxoglutarate to 1 eq. The chemical mechanism of glutamate synthase is very complex and still poorly understood. The Bacillus subtilis gene purF encoding amidophosphoribosyltransferase was cloned in E.
coli. The gene was expressed from a plasmid promoter and the plasmid-encoded enzyme functioned in E. coli. The nucleotide sequence of the gene was determined and yielded the derived amino acid sequence of the enzyme. The enzymes from these two bacteria are homologous. In contrast to the metalfree E. coli amidophosphoribosyl- transferase, the B. subtilis enzyme is an Fe-S protein. The reaction of glutamine was identified as the NH(2)-terminal residue of the mature enzyme.
Impacts
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Publications
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Progress 01/01/82 to 12/30/82
Outputs
Amidophosphoribosyltransferase is a key regulatory enzyme in the pathway for purine nucleotide synthesis. Amidophosphoribosyltransferase is one of approximately 10-15 glutamine amidotransferases, enzymes that utilize the amide of glutamine for incorporation of a nitrogen atom into biosynthetic products. Our previous work has concerned the purification and characterization of amidophosphoribosyltransferase from E. Coli. We have now cloned the E. Coli gene purF which encodes amidophosphoribosyltransferase. The nucleotide sequence of a 2478-base pair fragment encoding purF was determined. The structural gene codes for a 56,395 M(r) protein chain having 504 residues. The deduced amino acid sequence was confirmed by comparisons with the NH(2)-terminal amino acid sequence determined by automated Edman degradation and amino acid analyses of CNBr peptides obtained from the enzyme. Nucleotide sequences characteristic of bacterial promoter-operator regions were identified
in the 5' flanking region. Plasmid-bearing strains overproduce the enzyme thus facilitating enzyme purification. Purified E. Coli amidophosphoribosyltransferase lacks iron as well as other trace metals as determined by x-ray fluorescence spectrometry. This result indicates that in contrast to amidophosphoribosyltransferase from higher animals, the E. Coli enzyme lacks an FeS center. A role for FeS in catalysis can thus be eliminated.
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Progress 01/01/81 to 12/30/81
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Anthranilate synthase is the key regulatory enzyme in the pathway for tryptophansynthesis. The enzyme contains dissimilar subunits designated anthranilate synthase components I (AS I) and II (AS II). Our objective has been to determine the roles in catalysis of each of the subunits. Previous studies have defined the role of the active site cysteine in AS I. We have now employed techniques of chemical modification to probe for amino acid residues essential for AS I activity. Phenylglyoxal and 1,2-cyclohexanedione modified 2-5 arginine residues and inactivated AS I. Analysis of inactivation data indicated that 1 arginine residue is essential for activity. Histidine residues in AS I were modified by ethoxyformic anhydride and by photoxidation. Enzyme inactivation accompanied mofification of 1 histidine residue. Bromopyruvate inactivated AS I by allylation of 1 cysteine residue. The position of the cysteine was localized in a tryptic heptapeptide. Plausible roles
were suggested for the enzymes essential arginine, histidine and cysteine residures. The arginine residue may function in binding the anionic substrate, chorismic acid. Histidine is a good candidate for an acid-base group required for proton abstraction and donation. The essential cysteine residue may participate in covalent catalysis.
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Progress 01/01/80 to 12/30/80
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L-(Alpha S,5S)-Alpha-Amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125),an antitumor drug isolated from Streptomyces sviceus, is an active site-directed affinity analog of glutamine. It inactivates the glutamine-dependent activities of two bacterial glutamine amidotransferases, anthranilate synthase and glutamate synthase. A reversible non-covalent complex is formed prior to irreversible enzyme modification. Inactivation of anthranilate synthase results from incorporation of approximately 1 eq of AT-125/enzyme protomer. Active site cysteine-83 in Serratia marcescens anthranilate synthase Component II is the residue alkylated by AT-125. Anthranilate synthase is rapidly inactivated by AT-125 in S. marcescens cells. In vivo inactivation is by the same mechanism as in vitro. These experiments establish the mode of action of this drug whose use in humans is now under investigation. The drug is also a valuable probe for the active sites of glutamine
amidotransferases.
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Progress 01/01/79 to 12/30/79
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Glutamine phosphoribosylpyrophosphate amidotransferase (amidophosphoribosyltransferase) was purified to homogeneity from Escherichia coli and characterized. This enzyme catalyzes the first reaction of purine biosynthesis and is thus an important control site for regulating the levels of adenosine and guanosine nucleotides in cells. This enzyme has previously been refractory to purification from prokaryotic and eukaryotic sources. The E. coli enzyme is an oligomer of 3 or 4 identical chains each having a molecular weight of 57,000. In contrast to amidophosphoribosyltransferase from other sources, the E. coli enzyme lacks non heme iron. E. coli amidophosphoribosyltransferase was inhibited synergistically by AMP and GMP. Glutamine affinity analogs alkylated a single residue on each subunit, probably a cysteine, and inactivated the glutamine-dependent activity but not the NH(3)-dependent activity. Detailed studies on the catalytic and control mechanisms will require
larger amounts of homogeneous enzyme. Secretion of Alpha-amylase was studied in Bacilluys subtilis. Secreted enzyme, the product of AmyE, was in two forms, soluble and membrane bound. Each form was characterized. The soluable enzyme is a single polypeptide chain of molecular weight 67,000. The membrane-derived enzyme is immunologically similar but contains tightly associated phospholipid.
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Progress 01/01/78 to 12/30/78
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All living cell utilize ammonia as a source of nitrogen for the synthesis of amino acids, nucleic acids, vitamins and certain carbohydrates. The key steps in using ammonia are incorporation of inorganic ammonia into an organic form, glutamine and then transfer of the ammonia from glutamine to a large number of different precursor molecules to yield the required amino acids, etc. The latter steps in which the ammonia in glutamine is used for biosynthesis are catalyzed by a number of enzymes known as glutamine amidotransferases. This research is concerned with understanding the mechanisms by which glutamine amidotransferases function in the synthesis of amino acids and nucleic acids. In order to understand how an enzyme functions we need to understand its structure. We determine the amino acid sequence of one protein component of anthranilate synthase, the glutamine amidotransferase that functions to synthesize the amino acid tryptophan. We identified the region in
the enzyme known as the active site, the site where the reactants bind and are converted to the products. This is the first reported amino acid sequence of a glutamine amidotransferase. We also determined the amino acid sequence of a small segment of the active site region of a second amidotransferase, GMP synthetase. There was no amino acid sequence similarity in the active site regions of these two enzymes that were compared. This suggests that they function in somewhat different ways.
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Progress 01/01/77 to 12/30/77
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Characterization of E. coli GMP synthetase was indicated because this enzyme is representative of a group of glutamine amidotransferases in which NH(3)-dependent and glutamine-dependent activities are on a single protein chain. This structure-function relationship is in contrast to a group of glutamine amidotransferases, containing dissimilar subunits, in which the NH(3)-dependent reaction is catalyzed by one subunit and glutamine binding is by the other subunit. Glutamine affinity analogs, 6-diazo-5-oxonorleucine (DON) and 2-amino-4-oxo-5-chloropentanoic acid (CK) inactivated the glutamine-dependent activity, while the NH(3)-dependent activity was relatively unaffected, thus the sites for the two activities are distinct. Inactivation was accompanied by covalent attachment of 0.5 eq. of CK derivative. The alkylation reaction with DON and CK was mimicked by iodoacetamide. The stoichiometry of alkylation of iodoacetamide was 1 eq per subunit. Following acid
hydrolysis of iodoacetamide-treated enzyme only carboxy-methylcysteine was isolated. Thus similar to other glutamine amidotransferases containing non-identical subunits, glutamine appears to form a covalent enzyme intermediate, transfer the amide to the NH(3) site and form a covalent Gamma-glutamyl enzyme intermediate which is then hydrolyzed to yield glutamate. Amino acid sequence determination of a large tryptic peptide containing the "essential" cysteine residue is in progress.
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Progress 01/01/76 to 12/30/76
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This research is concerned with the mechanisms for utilization of NH(3) for biosynthesis of cell constituents. The synthesis of glutamine provides a major route for NH(3) assimilation. A group of 13 enzymes, designated glutamine amidotransferases, utilize the amide of glutamine' for biosynthesis of amino acids, nucleic acids and vitamins. Our objective is to study the mechanisms by which glutamine amidotransferases utilize the amide of glutamine. Two glutamine amidotransferases, anthranilate synthetase and glutamate synthase, involved in the synthesis of tryptophan and glutamate, respectively were characterized. Binding of glutamine and glutamine analogs to one protein chain of anthranilate synthetase (AS11) was studied. Evidence was obtained for formation of a covalent glutamyl-enzyme thioester intermediate. The amino acid sequence of the first 45 residues of AS II was determined. Work is in progress to complete the primary structure of this protein of
approximately 155 amino acids. We will then compare the glutamine active site region of other glutamine amidotransferases with that of AS II. Catalytic properties of glutamate synthasewere investigated and compared with anthranilate synthetase. The results suggest that these two enzymes have grossly different mechanisms.
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Progress 01/01/75 to 12/30/75
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Previous work from this laboratory indicates that the mechanism of glutamine utilization by anthranilate synthetase (AS) involves amide transfer from glutamine attached to one subunit (AS II) to an ammonia site on distynyy aminasesubunit (AS II). This mechanism has suggested that primitive cells utilized a strictly ammonia-dependent AS for tryptophan synthesis and that evolution of AS II in different organisms was from a common ancestral gene. Immunological methods were used to investigate structural similarities of AS from different organisms. Cross reactivity of anti-Escherichia coli AS and anti-Serratia marcescens AS with the enzyme from these organisms and Klebsiella aerogenes, Salmonella typhimurium, Pseudomonas putida and Bacillus subtilis was measured using precipitation, neutralization and immunodiffusion methods. Structural relatedness of AS in these organisms was similar to that for another enzyme of tryptopan synthesis, tryptophan synthetase a protein.
This result is compatiblewith divergent evolution from a common ancestral gene but is not proof. Anti-AS I inhibits the ammonia dependent reaction by blocking AS I site and inhibits theglutamine-dependent reaction by preventing the delivery of the amide to the ammonia site. Anti-AS ii inhibits the glutamine-dependent reaction by preventing delivery of the amide to the ammonia site. These results support thepreviously determined mechanism for glutamine utilization.
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Progress 01/01/74 to 12/30/74
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Fundamental knowledge of enzyme regulation should enhance our knowledge of cell metabolism and provide a basis for understanding and curing diseases and should contribute to the "engineering" of better sources of food. Studies on the regulation of enzyme activity have concerned enzymes that utilize the amide of glutamine for biosynthetic reactions: R + glutamine - RNH(2) + glutamate. We have been examining our hypothesis that many, if not all, amidotransferases havea common mechanism for glutamine utilization as a result of evolution from a common ancestral gene. Studies have been in progress with 3 glutamine amidotransferases in order to examine the hypothesis and to learn about the mechanism for glutamine utilization. The 3 enzymes are PRPP amidotransferase, implicate in gout, glutamate synthase, a key enzyme required for glutamic acid synthesis and anthranilate synthetase, an enzyme required for tryptophan synthesis. Antibodies have been prepared to
anthranilate synthetase in order toexamine relatedness in different microbial species and relatedness to other amidotransferases. The preliminary findings suggest some structural relationship to other glutamine amidotransferases. Other experiments have been directed toward labeling the enzyme site that binds glutamine. The procedure has been to attach irreversibly a radioactive glutamine analog and then degrade the enzyme and isolate a small protein segment containing the radioactive analog.
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Progress 01/01/73 to 12/30/73
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Work was initiated of developing an in vitro system in which tryptophan (trp) operon transcription coupled to enzyme synthesis could be studied biochemically,thereby providing the opportunity to examine aspects of gene expression. DNA-dependent synthesis of trp operon mRNA and enzymes was studied in vitro using template DNA from various null set80trp and null set80-lambdatrp phage andenzymes from E. coli. In vitro transcription and coupled translation were similar to the processes as they occur in the cell. Experiments were conducted to determine if ribosomes discharge from polycistronic trp mRNA following translation termination at natural chain termination codons. Obligatory discharge and subsequent reattachment of ribosomes were shown to occur. Synthesis of trp mRNA and enzymes was repressed by crude trp repressor protein and tryptophan when transcription was initiated at the trp operon promoter but not when transcription was initiated at a nearby phage
promoter. A nonsense mutation that gave rise to polarity in vivo did not exhibit polarity in vitro indicating that certain required factors were missing from the reconstituted in vitro system.
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Progress 01/01/72 to 12/30/72
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Anthranilate synthetase in plants and microorganisms catalyzes the first reaction of tryptophan biosynthesis and accordingly is subject to feedback control by this amino acid end product. A study was made concerning the mechanism by which anthranilate synthetase is inhibited by tryptophan. The following evidence leads to the conclusion that inhibition of enzyme activity occurs when tryptophan binds to a regulatory site and maintains the enzyme in a conformation having poor affinity for substrates. (1) Tryptophan binding causesconformational alterations in the enzyme. (2) The enzyme binds one tryptophan per subunit at a site at least partially distinct from the catalytic site (3) Binding of tryptophan at the regulatory site excludes binding of substrates. Inthe catalytic mechanism for this enzyme, one substrate chorismate must bind before the second substrate glutamine can bind. It was found that chorismate binding causes a conformational change in the enzyme
which could facilitate binding of glutamine. These results contribute to an understanding of how regulatory enzymes function to control metabolism.
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Progress 01/01/71 to 12/30/71
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Anthranilate synthetase is the first enzyme in the pathway for trypothan synthesis and is subject to endproduct regulation by tryptophan. This enzyme catalyzes the reaction of "-NH(2)" (from NH(2) or glutamine) chorismate to yieldanthranilate plus pyruvate. Major aspects of the reaction mechanism and controlmechanism were elucidated. Anthranilate synthetase from bacteria is always oligomeric and contains non identical subunits. One subunit functions in catalysis of the reaction with NH(3) and contains the site for the regulatory effector, tryptophan. The other subunit binds glutamine and transfers NH(3) to the first subunit. In some bacteria (Salmonella typhimiurium) the glutamine binding protein is covalently joined to another enzyme of tryptophan biosynthesis, while in other bacteria (Serratia marcescens) it is free. Tryptophan functions to inhibit enzyme activity by preventing binding of chorismate to the enzyme. This represents one of the few instances in
which themechanism for feedback inhibition of a regulatory enzyme has been defined. Other work has progressed toward examining the mechanism for inhibition of the regulatory enzyme for phenylalanine synthesis (chorismate mutase-prephenate dehydratase) by phenylalanine.
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Progress 01/01/70 to 12/30/70
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Studies were conducted on anthranilate synthetase-PR transferase (ASase-PRTase),the feedback regulated enzyme aggregate that controls tryptophan synthesis is Salmonella typhimurium. The principal findings were: (1) The composition of the aggregate appears to be (ASase)(2) (PRTase)(2). (2) Asase is a single polypeptide chain of mol. wt. 64,000. (3) PRTase appears to be a single polypeptide chain of mol. wt. 64,000. (4) The ASase-PRTase aggregate has a mol.wt. of about 260,000. (5) The catalytic and regulatory properties of each isolated component differ from those of the aggregate. (6) Feedback regulation of both ASase and PRTase by tryptophan occurs with cooperative interactions onlyin the aggregated state indicating that cooperativity is due to subunit interactions. (7) A unique and potentially basic mechanism for glutamine- and NH(3)-dependent amidotransferases was discovered. Glutamine, a substrate for ASase, binds to PRTase and a glutaminase transfers the
amide to ASase where it participates in the catalytic reaction. It appears that all glutamine amidotransferases are oligomeric and contain one subunit with a catalytic site and a second subunit which is a glutamine binding protein that generates NH(3) via glutaminase to the catalytic site. These results are significant because they help to clarify basic mechanisms for enzyme catalysis and regulation of metabolism.
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Progress 01/01/69 to 12/30/69
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Bifunctional enzyme that regulates phenylalanine synthesis in Salmonella typhimurium was partially pruified, This enzyme converts chorismate to prephenate (chorismate mutase activity) and prephenate to Phenylpyruvate (prephenate dehydratase activity) and has been named chorismate mutase prephenate dehydratase (CM D). Kinetic properties of both activities conform tothe allosteric K system model of monod, Wyman and Changeux. Phenylalanine shiftsthe equiligrium in favor of dimer. A mutant CM D, is monomeric even in the presence of phenylalanine. Cooperative kinetics and feedbacj inhibition of native CM-PD result from dimerization caused by phenylalanine. Further exploration should allow a clear picture of how phenylalanine regulates CM-PD activity. Anthranilate-5 phosphoribosplpyrophosphate phosphoribosylatransferase(Pr transferase) activity when aggregated to active or inactive anthranilate synthetase, is subject to endproduct-mediated substrate inhibition.
Inhibition of Pr transferase activity by the substrates PRPP and anthranilate is observed in presence of tryptophan.
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Progress 01/01/68 to 12/30/68
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Anthranilate synthetase component I has been purified from Salmonella typhimurium. In contrast to the native enzyme which is aggregated with component II, anthranilate-5-phosphoribo-sylpyrophosphatephosphoribosyltransferase, component I is inactive with glutamine as amino donor for anthranilate synthesis. Component I utilizes NH(4)+. Glutamine and glutamate inhibit anthranilate synthesis and are competitive with NH(4)RG, suggesting that component I retains aglutamine binding site. Initial velocity and product inhibition patterns are linear and are consistent with a sequential mechanism. The enzyme retains sensitivity to end-product inhibition by tryptophan. Reciprocal plots for inhibition by tryptophan are linear and show that tryptophan is competitive withchorismate and noncompetitive with NH(4)RG. There are no indications for homotropic or heterotropic interactions. The enzyme is inactivated by p-mercuribenzoate and N-ethylmaleimide. Partial reversal of
p-mercuribenzoate inhibition by dithiothreitol indicates that one or more sulfhydryl groups are essential for activity. Component I is also rapidly inactivated by a substrate analog, bromopyruvate. It is postulated that a basic group on the enzyme, susceptible to alkylation by bromopyruvate, participates in the removal of a proton from chorismate. Gel filtration on Sephadex G-100 provides an estimate of 63,000 for the molecular weight of component I. The protein recombines with component II and reactivity with glutamine is restored.
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