Biochemical Characterization and Crystal Structure Determination of Human Heart Short Chain l-3-Hydroxyacyl-CoA Dehydrogenase Provide Insights into Catalytic Mechanism

Abstract

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-oxidation pathway. The homodimeric enzyme has been overexpressed in Escherichia coli, purified to homogeneity, and studied using biochemical and crystallographic techniques. The dissociation constants of NAD+ and NADH have been determined over a broad pH range and indicate that SCHAD binds reduced cofactor preferentially. Examination of apparent catalytic constants reveals that SCHAD displays optimal enzymatic activity near neutral pH, with catalytic efficiency diminishing rapidly toward pH extremes. The crystal structure of SCHAD complexed with NAD+ has been solved using multiwavelength anomalous diffraction techniques and a selenomethionine-substituted analogue of the enzyme. The subunit structure is comprised of two domains. The first domain is similar to other alpha/beta dinucleotide folds but includes an unusual helix-turn-helix motif which extends from the central beta-sheet. The second, or C-terminal, domain is primarily alpha-helical and mediates subunit dimerization and, presumably, L-3-hydroxyacyl-CoA binding. Molecular modeling 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 proton 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 interaction with Glu 170, consistent with previous biochemical observations. These studies provide further understanding of the molecular basis of several inherited metabolic disease states correlated with L-3-hydroxyacyl-CoA dehydrogenase deficiencies.

Sequestration of the active site by interdomain shifting: crystallographic and spectroscopic evidence for distinct conformations of L-3-Hydroxyacyl-CoA dehydrogenase

“…Each model maintains the overall two domain topology of the previously described HAD-NAD + complex (2,17). The N-terminal domain of the monomer (residues 12-200) has a β-α-β fold similar to NAD(P) + -binding enzymes, and the C-terminal domain (residues 201-302) consists primarily of α-helices involved in subunit dimerization.…”

“…Initially, HAD was expressed with a C-terminal six-histidine tag using pET21a vector (Novagen) and purified by affinity chromatography as described previously (2). This construct contains a spurious mutation that results in the substitution of Cys for Phe at residue 80.…”

“…Crystals of human L-3-hydroxyacyl-CoA dehydrogenase were grown by the hanging drop method out of a solution of 50 mM N-[2-acetamido]-2-iminodiacetic acid, pH 6.5, within the precipitant range of 14% to 19% polyethylene glycol 4000 at a protein concentration of 5 mg/mL as described previously (2). In order to collect data at cryogenic temperatures, crystals were transferred to artificial mother liquor containing cryoprotectant (11).…”

“…In a previous report from this laboratory, the x-ray crystal structure of human L-3-hydroxyacyl-CoA dehydrogenase complexed with NAD + was described (2). The subunits of the dimeric enzyme are comprised of two domains.…”

The Multifunctional Protein in Peroxisomal β-Oxidation

“…3A). Amino acid residues observed to hydrogen bond to the cofactor in monofunctional HACDs (67,68) and PfMFP are conserved and have similar conformations in AtMFP2-HACD (Glu 342 , Glu 401 , Lys 406 , and Asn 624 ), but no significant electron density is observed in the AtMFP2 co-factor-binding site. A similar absence of positive electron density for the co-factor is observed in the crystal structure of the truncated recombinant RnMFE-1-HACD (15), whereas NAD ϩ is present in the active site of the PfMFP ␤-oxidation complex but with an average B-factor of 98 Å 2 (13).…”