The overall objective of this research is to extend our knowledge of structure-function relationships in thiamin diphosphate dependent alpha-keto acid decarboxylases; a large and ubiquitous class of enzymes of fundamental importance to basic metabolism. The project focuses on thiamin diphosphate dependent enzymes thal operate within large multienzyme complexes, and in particular, the E1 component of the pyruvate dehydrogenase multienzyme complex (PDHc). Such multienzyme complexes employ substrate channeling as a means of obtaining high efficiency, and few details are known regarding the key required interactions. The target E1 component from the E. coli PDHc complex is a member of the bacterial alpha (2) E1 family, and is highly homologous in sequence with its counterparts in many pathogenic organisms. The broad, long-term objective is to determine, analyze and understand the structure and function of an intact PDHc complex, while the short term objective is to probe the basic E1 reaction and its interactions with the E2 component.
The specific aims are: (1) To analyze protein-ligand complexes containing the PDHc E1 enzyme along with inhibitors, substrates and/or effectors. This includes the catalytic site directed inhibitors thiaminthiothiazolone diphosphate (ThTTDP), methyl acetylphosphinate, 2-oxo-3-butynoic acid, and fluropyruvate; the substrate/acceptor-substrate analogs pyruvate, lipoic acid and epsilon-N-Lipoyllysine (lipoamide); and the effectors guanosine triphosphate (GTP), acetyI-CoAJCoA and NAD+/NADH. (2) To obtain structure-function information for the E. coli apo-E1 enzyme and for other (non-E1) enzymatic components in the PDHc multienzyme complex, and to determine sites of protein-protein interaction required for complex assembly/function. (3) To identify structural effects resulting from single residue mutations in E. coli PDHc E1 at key catalytic site locations. (4) to extend structure-function analysis of PDHc interactions to include related enzymes from other sources, in particular, the PDHc complex from mycobacterium tuberculosis. The major method to be employed involves x-ray crystallographic studies of isolated proteins, protein-ligand complexes and protein-protein complexes.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM061791-05
Application #
6822961
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Program Officer
Preusch, Peter C
Project Start
2000-09-01
Project End
2008-08-31
Budget Start
2004-09-01
Budget End
2005-08-31
Support Year
5
Fiscal Year
2004
Total Cost
$210,870
Indirect Cost
Name
University of Pittsburgh
Department
Pharmacology
Type
Schools of Medicine
DUNS #
004514360
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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