STRUCTURAL BASES FOR CATALYTIC EFFICIENCY AND SUBSTRATE SPECIFICITY

Summary

Principal Investigator: JOHN GERLT
Affiliation: University of Illinois
Country: USA
Abstract: DESCRIPTION (provided by applicant): Structure-based explanations for the catalytic efficiencies and substrate specificities of enzymes are elusive. The goals of this project include 1) providing a structure-based explanation for the extraordinary catalytic efficiency of orotidine 5'-monophosphate decarboxylase (OMPDC) that catalyzes the final step in the pathway for pyrimidine nucleotide biosynthesis, and 2) characterizing the substrate specificity and mechanistic diversity in the D-ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) superfamily. Specific Aim 1 focuses on the roles of active site residues in both ground state destabilization (GSD) and transition state stabilization (TSS) in the OMPDC-catalyzed reaction. We have established that the reaction involves an intermediate, and we now want to understand the mechanisms by which the intermediate is generated and stabilized. We will examine the roles of specific amino acid residues in stabilizing negative charge that is delocalized in the pyrimidine ring of the intermediate (TSS) as well as those putatively involved in destabilizing the substrate carboxylate group (GSD). We will collaborate with Drs. Arthur Palmer, Columbia, for NMR studies of structure and dynamics and Dr. Jiali Gao, Minnesota, for computational studies. Specific Aim 2 focuses on the structural mechanism by which substrate binding is coupled to catalysis in the OMPDC-catalyzed reaction. Studies using both site-directed mutagenesis and "two part" substrate analogs (phosphite anion and a truncated orotidine, Drs. John Richard and Tina Amyes, Buffalo) demonstrate that the remote phosphate group synergizes decarboxylation. We will examine the conformational changes that accompany substrate binding to establish the roles of specific residues in coupling binding to catalysis. Specific Aim 3 is focused on characterizing mechanistic diversity in the RuBisCO superfamily. The structures suggest that the uncharacterized members will be excellent candidates for functional prediction using an integrated experimental and computational approach that successfully identified novel reactions in the amidohydrolase and enolase superfamilies;we will collaborate with Dr. Matthew P. Jacobson, UCSF, for both homology modeling and in silico library docking. As new functions are discovered, the mechanisms will be characterized so that the structural bases of functional and mechanistic diversity can be described. These studies will contribute to 1) delineating structural strategies for the design of inhibitors of OMPDC (beyond the scope of the current project);and 2) enhancing integrated structure-function-computation approaches for assigning the functions of uncharacterized proteins discovered in genome projects. PUBLIC HEALTH RELEVANCE: This project is focused on the important biomedical problem of exploiting genomic information to both assign functions to proteins discovered in genome sequencing projects and, also, to establish the structural bases for the biological functions. If new targets and experimental approaches are to be devised for small molecule intervention (drugs), the roles of all proteins involved in an organism's molecular, cellular, and organismal functions must be known. This project uses orotidine 5'-monophosphate decarboxylase (OMPDC), an essential enzyme in the biosynthesis of RNA and DNA, and homologues of D-ribulose 1,5-bisphosphate carbxoylase/oxygenase (RuBisCO), the ubiquitious enzyme that fixes CO2, to develop new knowledge about the structural basis for biological function. Our studies of OMPDC-catalyzed reaction will enable new strategies for the design of inhibitors for use as antibiotics;our studies of the RuBisCO superfamily will enhance the development of new approaches for assigning the functions of uncharacterized proteins discovered in genome projects.
Funding Period: ----------------2003 - ---------------2012-
more information: NIH RePORT

Top Publications

  1. ncbi D-Ribulose 5-phosphate 3-epimerase: functional and structural relationships to members of the ribulose-phosphate binding (beta/alpha)8-barrel superfamily
    Julie Akana
    Department of Biochemistry, University of Illinois at Urbana Champaign, 600 S Mathews Avenue, Urbana, Illinois 61801, USA
    Biochemistry 45:2493-503. 2006
  2. pmc Computational, structural, and kinetic evidence that Vibrio vulnificus FrsA is not a cofactor-independent pyruvate decarboxylase
    Whitney F Kellett
    Department of Chemistry, University of Florida, Gainesville, Florida 32611, United States
    Biochemistry 52:1842-4. 2013
  3. pmc Mechanistic diversity in the RuBisCO superfamily: RuBisCO from Rhodospirillum rubrum is not promiscuous for reactions catalyzed by RuBisCO-like proteins
    Benjamin P E Warlick
    Department of Biochemistry, University of Illinois at Urbana Champaign, Urbana, IL 61801, USA
    Biochemistry 51:9470-9. 2012
  4. pmc A RubisCO-like protein links SAM metabolism with isoprenoid biosynthesis
    Tobias J Erb
    Institute for Genomic Biology, University of Illinois at Urbana Champaign, Champaign, IL, USA
    Nat Chem Biol 8:926-32. 2012
  5. pmc 1-methylthio-D-xylulose 5-phosphate methylsulfurylase: a novel route to 1-deoxy-D-xylulose 5-phosphate in Rhodospirillum rubrum
    Benjamin P Warlick
    Department of Biochemistry, University of Illinois at Urbana Champaign, Urbana, IL 61801, USA
    Biochemistry 51:8324-6. 2012
  6. pmc Conformational changes in orotidine 5'-monophosphate decarboxylase: a structure-based explanation for how the 5'-phosphate group activates the enzyme
    Bijoy J Desai
    Departments of Biochemistry and Chemistry, University of Illinois at Urbana Champaign, Urbana, IL 61801, USA
    Biochemistry 51:8665-78. 2012
  7. pmc Proton transfer from C-6 of uridine 5'-monophosphate catalyzed by orotidine 5'-monophosphate decarboxylase: formation and stability of a vinyl carbanion intermediate and the effect of a 5-fluoro substituent
    Wing Yin Tsang
    Department of Chemistry, University at Buffalo, Buffalo, New York 14260, USA
    J Am Chem Soc 134:14580-94. 2012
  8. pmc Orotidine 5'-monophosphate decarboxylase: transition state stabilization from remote protein-phosphodianion interactions
    Tina L Amyes
    Department of Chemistry, University at Buffalo, Buffalo, NY 14260, USA
    Biochemistry 51:4630-2. 2012
  9. pmc Role of a guanidinium cation-phosphodianion pair in stabilizing the vinyl carbanion intermediate of orotidine 5'-phosphate decarboxylase-catalyzed reactions
    Bogdana Goryanova
    Department of Chemistry, University at Buffalo, Buffalo, New York 14260, United States
    Biochemistry 52:7500-11. 2013
  10. ncbi Evolution of enzyme superfamilies
    Margaret E Glasner
    Department of Biopharmaceutical Sciences, University of California, San Francisco, CA 94143, USA
    Curr Opin Chem Biol 10:492-7. 2006

Scientific Experts

  • JOHN GERLT
  • Chaya S Rapp
  • Tina L Amyes
  • John P Richard
  • B McKay Wood
  • Steven C Almo
  • Alexander A Fedorov
  • Heidi J Imker
  • Elena V Fedorov
  • Krisztina Toth
  • Kui K Chan
  • Bogdana Goryanova
  • Bijoy J Desai
  • Benjamin P Warlick
  • F Robert Tabita
  • Shonoi A Barnett
  • Bryant M Wood
  • Tobias J Erb
  • Jaya Sriram
  • Margaret E Glasner
  • Kui Chan
  • Patricia C Babbitt
  • Whitney F Kellett
  • Lawrence M Goldman
  • Wing Yin Tsang
  • Benjamin P E Warlick
  • Bradley S Evans
  • Vanessa Iiams
  • Julie Akana
  • Ursula Rothlisberger
  • Elizabeth Brunk
  • Nigel G J Richards
  • Weiming Wu
  • Jonathan S Sweedler
  • Udipi A Ramagopal
  • Jeffrey B Bonanno
  • Jonathan V Sweedler
  • Shonoi A Ming
  • Stephen K Burley
  • Kyuil Cho
  • Freeman M Wong
  • J Michael Sauder
  • Andrew Shabila
  • Jaya Singh
  • Walter R P Novak
  • Elena Fedorov

Detail Information

Publications27

  1. ncbi D-Ribulose 5-phosphate 3-epimerase: functional and structural relationships to members of the ribulose-phosphate binding (beta/alpha)8-barrel superfamily
    Julie Akana
    Department of Biochemistry, University of Illinois at Urbana Champaign, 600 S Mathews Avenue, Urbana, Illinois 61801, USA
    Biochemistry 45:2493-503. 2006
    ..Instead, this "superfamily" may result from assembly from smaller modules, including the conserved phosphate binding motif associated with the C-terminal (beta/alpha)(2)-quarter barrel...
  2. pmc Computational, structural, and kinetic evidence that Vibrio vulnificus FrsA is not a cofactor-independent pyruvate decarboxylase
    Whitney F Kellett
    Department of Chemistry, University of Florida, Gainesville, Florida 32611, United States
    Biochemistry 52:1842-4. 2013
    ..These results suggest that the functional annotation of FrsA as a cofactor-independent pyruvate decarboxylase is incorrect...
  3. pmc Mechanistic diversity in the RuBisCO superfamily: RuBisCO from Rhodospirillum rubrum is not promiscuous for reactions catalyzed by RuBisCO-like proteins
    Benjamin P E Warlick
    Department of Biochemistry, University of Illinois at Urbana Champaign, Urbana, IL 61801, USA
    Biochemistry 51:9470-9. 2012
    ..Thus, although RuBisCOs can be functionally promiscuous, RuBisCO from R. rubrum is not promiscuous for either of the known RLP-catalyzed reactions...
  4. pmc A RubisCO-like protein links SAM metabolism with isoprenoid biosynthesis
    Tobias J Erb
    Institute for Genomic Biology, University of Illinois at Urbana Champaign, Champaign, IL, USA
    Nat Chem Biol 8:926-32. 2012
    ....
  5. pmc 1-methylthio-D-xylulose 5-phosphate methylsulfurylase: a novel route to 1-deoxy-D-xylulose 5-phosphate in Rhodospirillum rubrum
    Benjamin P Warlick
    Department of Biochemistry, University of Illinois at Urbana Champaign, Urbana, IL 61801, USA
    Biochemistry 51:8324-6. 2012
    ..et al. (2012) Nat. Chem. Biol., in press]. Dethiomethylation, a novel route to DXP, is catalyzed by MTXu 5-P methylsulfurylase. An active site Cys displaces the enolate of DXP from MTXu 5-P, generating a methyl disulfide intermediate...
  6. pmc Conformational changes in orotidine 5'-monophosphate decarboxylase: a structure-based explanation for how the 5'-phosphate group activates the enzyme
    Bijoy J Desai
    Departments of Biochemistry and Chemistry, University of Illinois at Urbana Champaign, Urbana, IL 61801, USA
    Biochemistry 51:8665-78. 2012
    ....
  7. pmc Proton transfer from C-6 of uridine 5'-monophosphate catalyzed by orotidine 5'-monophosphate decarboxylase: formation and stability of a vinyl carbanion intermediate and the effect of a 5-fluoro substituent
    Wing Yin Tsang
    Department of Chemistry, University at Buffalo, Buffalo, New York 14260, USA
    J Am Chem Soc 134:14580-94. 2012
    ....
  8. pmc Orotidine 5'-monophosphate decarboxylase: transition state stabilization from remote protein-phosphodianion interactions
    Tina L Amyes
    Department of Chemistry, University at Buffalo, Buffalo, NY 14260, USA
    Biochemistry 51:4630-2. 2012
    ..The results are consistent with a model in which phosphodianion binding interactions are utilized to stabilize a rare closed enzyme form that exhibits a high catalytic activity for decarboxylation...
  9. pmc Role of a guanidinium cation-phosphodianion pair in stabilizing the vinyl carbanion intermediate of orotidine 5'-phosphate decarboxylase-catalyzed reactions
    Bogdana Goryanova
    Department of Chemistry, University at Buffalo, Buffalo, New York 14260, United States
    Biochemistry 52:7500-11. 2013
    ....
  10. ncbi Evolution of enzyme superfamilies
    Margaret E Glasner
    Department of Biopharmaceutical Sciences, University of California, San Francisco, CA 94143, USA
    Curr Opin Chem Biol 10:492-7. 2006
    ..Understanding how enzyme superfamilies evolve is vital for accurate genome annotation, predicting protein functions, and protein engineering...
  11. ncbi Mechanisms of protein evolution and their application to protein engineering
    Margaret E Glasner
    Department of Biopharmaceutical Sciences, University of California San Francisco, San Francisco, CA 94143, USA
    Adv Enzymol Relat Areas Mol Biol 75:193-239, xii-xiii. 2007
    ..Future research will determine if application of these principles will lead to a protein engineering methodology governed by predictable rules for designing efficient, novel catalysts...
  12. ncbi Mechanistic diversity in the RuBisCO superfamily: the "enolase" in the methionine salvage pathway in Geobacillus kaustophilus
    Heidi J Imker
    Departments of Biochemistry and Chemistry, University of Illinois, 600 South Mathews Avenue, Urbana, Illinois 61801, USA
    Biochemistry 46:4077-89. 2007
    ..e., coordination to an essential Mg2+, but the proton abstraction is catalyzed by a different general base...
  13. pmc Mechanistic diversity in the RuBisCO superfamily: a novel isomerization reaction catalyzed by the RuBisCO-like protein from Rhodospirillum rubrum
    Heidi J Imker
    Department of Microbiology and Plant Cellular and Molecular Biology Department Plant Molecular Biology Biotechnology Program, The Ohio State University, 484 West 12th Avenue, Columbus, Ohio 43210, USA
    Biochemistry 47:11171-3. 2008
    ..Disruption of the gene encoding the RLP abolishes the ability of R. rubrum to utilize 5'-methylthioadenosine as a sole sulfur source, implicating a new, as-yet-uncharacterized, pathway for sulfur salvage...
  14. pmc Product deuterium isotope effect for orotidine 5'-monophosphate decarboxylase: evidence for the existence of a short-lived carbanion intermediate
    Krisztina Toth
    Department of Chemistry, University at Buffalo, Buffalo, New York 14260, USA
    J Am Chem Soc 129:12946-7. 2007
  15. pmc Formation and stability of a vinyl carbanion at the active site of orotidine 5'-monophosphate decarboxylase: pKa of the C-6 proton of enzyme-bound UMP
    Tina L Amyes
    Department of Chemistry, University at Buffalo, Buffalo, New York 14260, USA
    J Am Chem Soc 130:1574-5. 2008
  16. pmc Dissecting the total transition state stabilization provided by amino acid side chains at orotidine 5'-monophosphate decarboxylase: a two-part substrate approach
    Shonoi A Barnett
    Department of Chemistry, University at Buffalo, SUNY, Buffalo, New York 14260 3000, USA
    Biochemistry 47:7785-7. 2008
    ....
  17. pmc Structural basis for substrate specificity in phosphate binding (beta/alpha)8-barrels: D-allulose 6-phosphate 3-epimerase from Escherichia coli K-12
    Kui K Chan
    Departments of Biochemistry and Chemistry, University of Illinois at Urbana Champaign, 600 South Mathews Avenue, Urbana, Illinois 61801, USA
    Biochemistry 47:9608-17. 2008
    ....
  18. pmc Enzyme (re)design: lessons from natural evolution and computation
    John A Gerlt
    Departments of Biochemistry and Chemistry, University of Illinois, Urbana, 61801, United States
    Curr Opin Chem Biol 13:10-8. 2009
    ..This review summarizes recent progress in evolution-based and computation-based (re)design...
  19. pmc Mechanism of the orotidine 5'-monophosphate decarboxylase-catalyzed reaction: importance of residues in the orotate binding site
    Vanessa Iiams
    Departments of Biochemistry and Chemistry, University of Illinois, Urbana, Illinois 61801, United States
    Biochemistry 50:8497-507. 2011
    ..These observations provide additional information about the identities of the active site residues that contribute to the rate enhancement and, therefore, insights into the structural strategies for catalysis...
  20. pmc OMP decarboxylase: phosphodianion binding energy is used to stabilize a vinyl carbanion intermediate
    Bogdana Goryanova
    Department of Chemistry, University at Buffalo, Buffalo, New York 14260, USA
    J Am Chem Soc 133:6545-8. 2011
    ..These results show that the intrinsic binding energy of phosphite dianion is used in the stabilization of the vinyl carbanion-like transition state common to the decarboxylation and deuterium exchange reactions...
  21. pmc Product deuterium isotope effects for orotidine 5'-monophosphate decarboxylase: effect of changing substrate and enzyme structure on the partitioning of the vinyl carbanion reaction intermediate
    Krisztina Toth
    Department of Chemistry, University at Buffalo, Buffalo, New York 14260, USA
    J Am Chem Soc 132:7018-24. 2010
    ..Mutations on the following important active-site residues of OMPDC from S. cerevisiae have no effect on the PIE on OMPDC-catalyzed decarboxylation of OMP or decarboxylation of 5-FOMP: R235A, Y217A, Q215A, S124A, and S154A/Q215A...
  22. pmc Conformational changes in orotidine 5'-monophosphate decarboxylase: "remote" residues that stabilize the active conformation
    B McKay Wood
    Department of Biochemistry, University of Illinois, Urbana, Illinois 61801, USA
    Biochemistry 49:3514-6. 2010
    ..The intrinsic binding energies of the 5'-phosphate group of orotidine 5'-monophosphate for the mutant enzymes are similar to that for the wild type, supporting this conclusion...
  23. pmc Activation of R235A mutant orotidine 5'-monophosphate decarboxylase by the guanidinium cation: effective molarity of the cationic side chain of Arg-235
    Shonoi A Barnett
    Department of Chemistry, University at Buffalo, SUNY, Buffalo, New York 14260 3000, USA
    Biochemistry 49:824-6. 2010
    ..The "effective molarity" of the cationic side chain of Arg-235 at the wild-type enzyme is calculated to be 160 M...
  24. pmc Mechanism of the orotidine 5'-monophosphate decarboxylase-catalyzed reaction: evidence for substrate destabilization
    Kui K Chan
    Department of Biochemistry, University of Illinois at Urbana Champaign, Urbana, Illinois 61801, USA
    Biochemistry 48:5518-31. 2009
    ..We propose that the differential effects of substitutions for Asp 70 of MtOMPDC on decarboxylation and exchange provide additional evidence for a carbanionic intermediate as well as the involvement of Asp 70 in substrate destabilization...
  25. pmc Mechanism of the orotidine 5'-monophosphate decarboxylase-catalyzed reaction: effect of solvent viscosity on kinetic constants
    B McKay Wood
    Department of Biochemistry, University of Illinois at Urbana Champaign, Urbana, Illinois 61801, USA
    Biochemistry 48:5510-7. 2009
    ..e., substrate binding and product dissociation mask the kinetic effects of stabilization of the intermediate by the substituent...
  26. pmc Automated site preparation in physics-based rescoring of receptor ligand complexes
    Chaya S Rapp
    Department of Chemistry, Stern College for Women, Yeshiva University, New York, New York 10016, USA
    Proteins 77:52-61. 2009
    ..7 to 1.5 A when applying ICDA. Large improvements are seen for specific classes of binding sites with titratable groups, such as aspartyl proteases...
  27. pmc An examination of the relationship between active site loop size and thermodynamic activation parameters for orotidine 5'-monophosphate decarboxylase from mesophilic and thermophilic organisms
    Krisztina Toth
    Department of Chemistry, University at Buffalo, SUNY, Buffalo, New York 14260 3000, USA
    Biochemistry 48:8006-13. 2009
    ....