Poster: Enzymology
Abs #
243: Phosphorylation of mitochondrial pyruvate dehydrogenase from Arabidopsis thaliana
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Presenter: |
Hirani, Tripty A., triptyhirani@hotmail.com |
Authors | Hirani, Tripty A. (A) Tovar-Mendez, Alejandro (A) Miernyk, Jan A. (A) (B) Randall, Douglas D. (A) | | Affiliations: |
(A): Department of Biochemistry, University of Missouri
(B): Plant Genetics Research Unit, USDA, Agricultural Research Service
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The pyruvate dehydrogenase complex (PDC) is a large multi-component structure that catalyzes oxidative decarboxylation of pyruvate yielding CO2, acetyl-CoA and NADH. The decarboxylation reaction is catalyzed by pyruvate dehydrogenase (E1), an a2b2 heterotetramer. Mitochondrial PDC is regulated by phosphorylation of E1 by the pyruvate dehydrogenase kinase (PDK) that inactivates the PDC. Regulation of mammalian E1 involves multi-site phosphorylation on E1a. Of these, site 1 is conserved in plants, site 2 being a Thr and site 3 an Ala residue. There is, however, a Ser residue immediately upstream of the Thr residue in plants that is the putative second site of phosphorylation. The coding sequences of E1a and E1b were cloned into pT7-7 and pET28 vectors, respectively, and coexpressed in E.coli BL21(DE3). A His6 tag was fused to the N-terminus of E1aa to allow for affinity purification using immobilized NiSO4. A series of site 1 and site 2 mutants in E1a were generated with the aim of investigating their roles in the phosphorylation of E1. These mutants comprised of S292A, S298A, S292A/S298A, S292D/S298A, and S292E/S298A. The S298A mutant was similar to wild-type E1 in its catalytic activity, assayed as decarboxylation of 14C-pyruvate, and its phosphorylation by the recombinant E1-kinase (r AtPDK), however, the S292A mutant was 60% active and could not be phosphorylated. The rest of the mutants were neither active nor phosphorylated. It is believed that interaction of E1 with the lipoyl domain of the dihydrolipoyl acetyltransferase exposes the second site for phosphorylation. Data will be presented on this interaction. Furthermore, the effect of the cofactor thiamine pyrophosphate on the phosphorylation site mutants will be presented.
