Biochemical characterization and crystal structure determination of human heart short chain L-3-Hydroxyacyl-CoA dehydrogenase provide insights into catalytic mechanism

Human heart short chain L-3-hydroxyacyl-CoA dehydrogenase(SCHAD) catalyzes the oxidation of the hydroxyl group of L-3-hydroxyacyl-CoA to a keto group, concomitant with the reduction of NAD(+) to NADH, as part of the beta-oxid ation pathway. The homodimeric enzyme has been overexpressed in Escherichia coli, purified to homogeneity, and studied using biochemical and crystallo graphic techniques. The dissociation constants of NAD(+) and NADH have been determined over a broad pH range and indicate that SCHAD binds reduced cof actor preferentially. Examination of apparent catalytic constants reveals t hat SCHAD displays optimal enzymatic activity near neutral pH, with catalyt ic efficiency diminishing rapidly toward pH extremes. The crystal structure of SCHAD complexed with NAD(+) has been solved using multiwavelength anoma lous diffraction techniques and a selenomethionine-substituted analogue of the enzyme. The subunit structure is comprised of two domains. The first do main is similar to other alpha/beta dinucleotide folds but includes an unus ual helix-turn-helix motif which extends from the central beta-sheet. The s econd, or C-terminal, domain is primarily alpha-helical and mediates subuni t dimerization and, presumably, L-3-hydroxyacyl-CoA binding. Molecular mode ling studies in which L-3-hydroxybutyryl-CoA was docked into the enzyme-NAD (+) complex suggest that His 158 serves as a general base, abstracting a pr oton from the 3-OH group of the substrate. Furthermore, the ability of His 158 to perform such a function may be enhanced by an electrostatic interact ion with Glu 170, consistent with previous biochemical observations. These studies provide further understanding of the molecular basis of several inh erited metabolic disease states correlated with L-3-hydroxyacyl-CoA dehydro genase deficiencies.