This is a request for renewal of a research program on thiamin diphosphate-dependent enzymes, principally on the E. coli and human pyruvate dehydrogenase multienzyme complexes. These complexes are at a key junction in metabolism of virtually all cells, converting the product of glycolysis pyruvic acid to acetyl coenzyme A at the entry to the Krebs cycle (also known as the tricarboxylic acid or citric acid cycle). Goals for the next phase of the project are: (1) Determination of rate-limiting steps, states of ionization and tautomerization of enzyme-bound thiamin-related intermediates, with the intent to compare these properties in isolated components of the complexes to those in the 4,600,000 and 10,000,000 Da complexes. In innovation requiring de novo total synthesis of specifically labeled thiamins, the PI proposes to use solid state NMR, as well as advanced solution NMR methods to accomplish the goals. (2) Exploration of the hypothesis that the mobility of active center loops in thiamin enzymes is correlated with catalysis. With methods published by the PI in 2008, key loops on the E. coli complex's E1 and E2 components have been identified for these studies, including the lipoyl domain of the E2 component, which 'visits'the active centers of all three components. (3) Examination of the structural and functional consequences of assembly in the bacterial and human pyruvate dehydrogenase complexes. In the E. coli complex, the issue to be resolved is whether the same region(s) of the E2 component recognizes the E1 and E3 components, to address the hypothesis that the loci of recognition of E2 for E1 or E3 are different. In the human complex, we wish to determine the regions of E2 component interacting with the E1 component, never accomplished before The methods used by the PI could answer questions that no other current methodology can, such as the definition of proton positions on the 4'-aminopyrimidine ring of the coenzyme at distinct intermediates along the reaction coordinate, a key issue in understanding proton transfers. A group of outstanding collaborators have been recruited for experimental expertise not available in the PI's laboratory, among them M. Patel, a CoPI selected for expertise on the human enzyme, and W. Furey, a long-term collaborator in a string of structure determinations. Results from two complexes will enable the PI to draw general conclusions regarding the entire superfamily of such enzymes, as already demonstrated in several of his recent publications.

Public Health Relevance

This is a request for renewal of a research program on thiamin diphosphate dependent enzymes, principally on the E. coli and human pyruvate dehydrogenase multienzyme complexes. These complexes are at a key junction in metabolism of virtually all cells, converting the product of glycolysis pyruvic acid to acetyl coenzyme A at the entry of the Krebs cycle (also known as the tricarboxylic acid or citric acid cycle). Goals for the next phase of the project include: (1) Determination of rate-limiting steps, states of ionization and tautomerization of enzyme-bound thiamin-related intermediates, with the intent to compare these properties in isolated components of the complexes to those in the 4,600,000 and 10,000,000 Da complexes. (2) Explore the hypothesis that the mobility of active center loops in thiamin enzymes is correlated with catalysis. (3) Examine the structural and functional consequences of assembly in the bacterial and human pyruvate dehydrogenase complexes. In the E. coli complex, the issue to be resolved is whether the same region(s) of the E2 component recognize the E1 and E3 components, to address the hypothesis that the loci of recognition of E2 for E1 or E3 are different. In the human complex, the issue to be addressed is determination of the regions of E2 component interacting with the E1 component, never accomplished before. Recent evidence indicates involvement of the kinase-dependent control of the human pyruvate dehydrogenase complex activity in both diabetes and cancer making structural studies of the human E2 component particularly timely. The studies proposed are intertwined with structural studies carried out in collaboration with highly productive and prominent groups.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM050380-15
Application #
8197484
Study Section
Macromolecular Structure and Function E Study Section (MSFE)
Program Officer
Gerratana, Barbara
Project Start
1996-05-01
Project End
2013-11-30
Budget Start
2011-12-01
Budget End
2012-11-30
Support Year
15
Fiscal Year
2012
Total Cost
$366,894
Indirect Cost
$129,294
Name
Rutgers University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
130029205
City
Newark
State
NJ
Country
United States
Zip Code
07102
Ambrus, Attila; Nemeria, Natalia S; Torocsik, Beata et al. (2015) Formation of reactive oxygen species by human and bacterial pyruvate and 2-oxoglutarate dehydrogenase multienzyme complexes reconstituted from recombinant components. Free Radic Biol Med 89:642-50
Arjunan, Palaniappa; Wang, Junjie; Nemeria, Natalia S et al. (2014) Novel binding motif and new flexibility revealed by structural analyses of a pyruvate dehydrogenase-dihydrolipoyl acetyltransferase subcomplex from the Escherichia coli pyruvate dehydrogenase multienzyme complex. J Biol Chem 289:30161-76
Patel, Mulchand S; Nemeria, Natalia S; Furey, William et al. (2014) The pyruvate dehydrogenase complexes: structure-based function and regulation. J Biol Chem 289:16615-23
Wang, Junjie; Nemeria, Natalia S; Chandrasekhar, Krishnamoorthy et al. (2014) Structure and function of the catalytic domain of the dihydrolipoyl acetyltransferase component in Escherichia coli pyruvate dehydrogenase complex. J Biol Chem 289:15215-30
Brammer Basta, Leighanne A; Patel, Hetalben; Kakalis, Lazaros et al. (2014) Defining critical residues for substrate binding to 1-deoxy-D-xylulose 5-phosphate synthase--active site substitutions stabilize the predecarboxylation intermediate C2α-lactylthiamin diphosphate. FEBS J 281:2820-37
Nemeria, Natalia S; Ambrus, Attila; Patel, Hetalben et al. (2014) Human 2-oxoglutarate dehydrogenase complex E1 component forms a thiamin-derived radical by aerobic oxidation of the enamine intermediate. J Biol Chem 289:29859-73
Patel, Hetalben; Shim, Da Jeong; Farinas, Edgardo T et al. (2013) Investigation of the donor and acceptor range for chiral carboligation catalyzed by the E1 component of the 2-oxoglutarate dehydrogenase complex. J Mol Catal B Enzym 98:
Jordan, Frank; Patel, Hetalben (2013) Catalysis in Enzymatic Decarboxylations: Comparison of Selected Cofactor-dependent and Cofactor-independent Examples. ACS Catal 3:1601-1617
Chandrasekhar, Krishnamoorthy; Wang, Junjie; Arjunan, Palaniappa et al. (2013) Insight to the interaction of the dihydrolipoamide acetyltransferase (E2) core with the peripheral components in the Escherichia coli pyruvate dehydrogenase complex via multifaceted structural approaches. J Biol Chem 288:15402-17
Balakrishnan, Anand; Jordan, Frank; Nathan, Carl F (2013) Influence of allosteric regulators on individual steps in the reaction catalyzed by Mycobacterium tuberculosis 2-hydroxy-3-oxoadipate synthase. J Biol Chem 288:21688-702

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