Structure and Function of the Catalytic Domain of the Dihydrolipoyl Acetyltransferase Component in Escherichia coli Pyruvate Dehydrogenase Complex [Metabolism]

April 17th, 2014 by Wang, J., Nemeria, N. S., Chandrasekhar, K., Kumaran, S., Arjunan, P., Reynolds, S., Calero, G., Brukh, R., Kakalis, L., Furey, W., Jordan, F.

The E. coli pyruvate dehydrogenase complex (PDHc) catalyzing conversion of pyruvate to acetyl-CoA comprises three components E1p, E2p and E3. The E2p is the five-domain core component, consisting of three tandem lipoyl domains (LD), a peripheral subunit binding domain (PSBD) and catalytic domain (E2pCD). Herein are reported: (1) The X-ray structure of E2pCD revealed both intra- and inter-trimer interactions, similar to those reported for other E2pCDs. (2) Reconstitution of recombinant LD, E2pCD with E1p, E3p to PDHc could maintain at least 6.4% activity (NADH production) confirming functional competence of the E2pCD and active center coupling among E1p, LD, E2pCD and E3 even in the absence of PSBD and of a covalent link between domains within E2p. (3) Direct acetyl transfer between LD and coenzymeA catalyzed by E2pCD was observed with a rate constant of 199 s-1, comparable to the rate of NADH production in the PDHc reaction. Hence, neither reductive acetylation of E2p, nor acetyl transfer within E2p is rate limiting. (4) An unprecedented finding is that although no interaction could be detected between E1p and E2pCD by itself, a domain-induced interaction was identified on E1p active centers upon assembly with E2p and C-terminally truncated E2p proteins by H/D exchange mass spectrometry. Inclusion of each additional domain of E2p strengthened the interaction with E1p, and was strongest with intact E2p. E2p domain-induced changes at the E1p active site were also manifest by the appearance of a circular dichroism band characteristic of the canonical 4-aminopyrimidine tautomer of bound thiamin diphosphate.
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