The overall objective of this research is to extend our knowledge of structure-function relationships in a large class of enzymes of fundamental importance to basic metablism; the thiamin diphosphate dependent alpha-keto acid decarboxylases. The project focuses on thiamin dependent enzymes that function within large multienzyme complexes, and their relationship to similar enzymes functioning in isolation. Despite their widespread importance in biochemistry and many years of study, only a single, recently obtained structural example exists for such a multienzyme complex components. Thus even the most basic structural information has just become available for any member of this family, and numerous questions remain regarding the catalytic, regulatory and assembly mechanisms. The goal is to provide the first high-resolution crystal structure analysis of a thiamin dependent E1 component from a pyruvate dehydrogenase multi-enzyme complex, PDHc E1 from E. coli, and compare it to other related enzymes. The structures it's to be compared with include that for pyruvate decarboxylase (PDC), which carries out essentially the same reaction on the same substrate but in isolation rather than in a complex, and that for the only other E1 structure for which data are available; the E1b component from a branched chain 2-oxoisovalerate dehydrogenase multienzyme complex. The latter (BDHcE1b) represents a different E1 class having no sequence homology, a different size and subunit composition, a different oligomeric state and operates on a different substrate than the E1 to be studied. X-ray diffraction methods also will be used to provide detailed information about structural changes resulting from interaction wth activators, substrates and inhibitors.
The specific aims are: (1) to determine and analyze the structure of PDHc E1 to a resolution suitable for a complete chain trace; (2) To refine the complete structure to the highest resolution possible; (3) To compare th PDHc E1 structure with that for PDC; (4) To compare the PDHc E1 structure with that for BDHc E1b; (5) To identify amino acids in the PDHc E1 active site, assign functional roles to each and identify residues involved in lipoamide binding; (6) To identify binding sites and inhibition mechanisms for fluropyruvate, 2-oxo-3-butynoic acid, thiamin-2-thiothiazolone and monoclonal antibody 18A9; (7) To identify regulatory binding sites for GTP, Acetyl-CoA/CoA and probe pathways for information transfer to catalytic sites; (8) To analyze structures of complexes with covalently bound reaction intermediate analogs (phosphonate or phosphinate analogs of pyruvate).

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM061791-02
Application #
6387227
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Program Officer
Ikeda, Richard A
Project Start
2000-09-01
Project End
2004-08-31
Budget Start
2001-09-01
Budget End
2002-08-31
Support Year
2
Fiscal Year
2001
Total Cost
$179,900
Indirect Cost
Name
University of Pittsburgh
Department
Pharmacology
Type
Schools of Medicine
DUNS #
053785812
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Whitley, Matthew J; Arjunan, Palaniappa; Nemeria, Natalia S et al. (2018) Pyruvate dehydrogenase complex deficiency is linked to regulatory loop disorder in the ?V138M variant of human pyruvate dehydrogenase. J Biol Chem 293:13204-13213
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
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
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
Nemeria, Natalia S; Arjunan, Palaniappa; Chandrasekhar, Krishnamoorthy et al. (2010) Communication between thiamin cofactors in the Escherichia coli pyruvate dehydrogenase complex E1 component active centers: evidence for a ""direct pathway"" between the 4'-aminopyrimidine N1' atoms. J Biol Chem 285:11197-209
Kale, Sachin; Ulas, Gozde; Song, Jaeyoung et al. (2008) Efficient coupling of catalysis and dynamics in the E1 component of Escherichia coli pyruvate dehydrogenase multienzyme complex. Proc Natl Acad Sci U S A 105:1158-63
Kale, Sachin; Arjunan, Palaniappa; Furey, William et al. (2007) A dynamic loop at the active center of the Escherichia coli pyruvate dehydrogenase complex E1 component modulates substrate utilization and chemical communication with the E2 component. J Biol Chem 282:28106-16
Chandrasekhar, Krishnamoorthy; Arjunan, Palaniappa; Sax, Martin et al. (2006) Active-site changes in the pyruvate dehydrogenase multienzyme complex E1 apoenzyme component from Escherichia coli observed at 2.32 A resolution. Acta Crystallogr D Biol Crystallogr 62:1382-6
Arjunan, Palaniappa; Sax, Martin; Brunskill, Andrew et al. (2006) A thiamin-bound, pre-decarboxylation reaction intermediate analogue in the pyruvate dehydrogenase E1 subunit induces large scale disorder-to-order transformations in the enzyme and reveals novel structural features in the covalently bound adduct. J Biol Chem 281:15296-303

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