Ligand-induced Conformational Changes and a Reaction Intermediate in Branched-chain 2-Oxo Acid Dehydrogenase (E1) from Thermus thermophilus HB8, as Revealed by X-ray Crystallography

The α2β2 tetrameric E1 component of the branched-chain 2-oxo acid (BCOA) dehydrogenase multienzyme complex is a thiamin diphosphate (ThDP)-dependent enzyme. E1 catalyzes the decarboxylation of a BCOA concomitant with the formation of the α-carbanion/enamine intermediate, 2-(1-hydroxyalkyl)-ThDP, followed by transfer of the 1-hydroxyalkyl group to the distal sulfur atom on the lipoamide of the E2 component. In order to elucidate the catalytic mechanism of E1, the α- and β-subunits of E1 from Thermus thermophilus HB8 have been co-expressed in Escherichia coli, purified and crystallized as a stable complex, and the following crystal structures have been analyzed: the apoenzyme (E1apo), the holoenzyme (E1holo), E1holo in complex with the substrate analogue 4-methylpentanoate (MPA) as an ES complex model, and E1holo in complex with 4-methyl-2-oxopentanoate (MOPA) as the α-carbanion/enamine intermediate (E1ceim). Binding of cofactors to E1apo induces a disorder–order transition in two loops adjacent to the active site. Furthermore, upon binding of MPA to E1holo, the loop comprised of Gly121β-Gln131β moves close to the active site and interacts with MPA. The carboxylate group of MPA is recognized mainly by Tyr86β and N4′ of ThDP. The hydrophobic moiety of MPA is recognized by Phe66α, Tyr95α, Met128α and His131α. As an intermediate, MOPA is decarboxylated and covalently linked to ThDP, and the conformation of the protein loop is almost the same as in the substrate-free (holoenzyme) form. These results suggest that E1 undergoes an open–closed conformational change upon formation of the ES complex with a BCOA, and the mobile region participates in the recognition of the carboxylate group of the BCOA. ES complex models of E1holo·MOPA and of E1ceim·lipoamide built from the above structures suggest that His273α and His129β′ are potential proton donors to the carbonyl group of a BCOA and to the proximal sulfur atom on the lipoamide, respectively.