L-3-Hydroxyacyl-CoA dehydrogenase (HAD), the penultimate enzyme in the ␤-oxidation spiral, reversibly catalyzes the conversion of L-3-hydroxyacyl-CoA to the corresponding 3-ketoacyl-CoA. L-3-Hydroxyacyl-CoA dehydrogenase (HAD 1 ; EC 1.1.1.35) is the penultimate enzyme in the ␤-oxidation spiral, reversibly catalyzing the oxidation of the hydroxyl group of L-3-hydroxyacyl-CoA to a keto group, concomitant with the reduction of NAD ϩ to NADH, as shown in Scheme 1. The dimeric enzyme displays broad substrate specificity, utilizing substrates with 4 -16 carbons in the acyl chain (1). Hydride transfer occurs at the pro-S position of the nicotinamide ring, making HAD a "B-side"-specific dehydrogenase (2 170 also forms contacts that may be important for dynamic enzyme movements. HAD exhibits a two-domain topology (7), with the N-terminal domain (residues 12-200) adopting a ␤-␣-␤ fold similar to NAD(P) ϩ -binding enzymes and the C-terminal domain (residues 201-302) consisting primarily of ␣-helices involved in subunit dimerization. Two distinct conformers of the enzyme structure have been identified (5,6). In the open conformation, as described for apo and cofactor-bound enzyme, a large cleft is observed between the NAD ϩ -binding and the C-terminal domains. The addition of substrate results in a conformational change in which the NAD ϩ -binding domain rotates inward toward the dimer interface, sequestering the enzyme active site. This domain shift appears necessary for high affinity substrate binding and critical for effective catalysis. The linker region, composed of residues 201-207, relates the two domains and contains a consensus Pro 203 -Gly 204 -Phe 205 sequence, which appears to be the pivot point for domain movement. Numerous interactions between Glu 170 and residues within this region are observed, including a hydrogen bond with a conserved water molecule.Charge transfer complex formation by HAD provides a spectroscopic assay for structural integrity that can be used to complement binding studies and kinetic analysis. As described previously, the abortive ternary complex composed of HAD, NAD ϩ , and AACoA exhibits a broad absorbance band centered between 410 and 420 nm (6). AACoA, which is bound as an enolate in the abortive complex, acts as an electron donating species and the nicotinamide ring of NAD ϩ serves as the electron acceptor. The intensity of the charge transfer band is pH-dependent, with the protonation of a single group resulting in decreased enolate and charge transfer complex formation. The spectroscopic properties of the charge transfer complex are sensitive to perturbations in the protein structure and can be used to probe active site integrity.To evaluate the contribution of Glu 170 to catalysis, this residue was substituted with glutamine by site-directed mutagenesis, and the resultant enzyme (E170Q) was analyzed by ki-* This work was supported by National Institutes of Health Grants 1F32-DK09759 (to J. J. B.) and GM13925 (to L. J. B.). The costs of publication of this article were defrayed in...

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Short chain L-3-hydroxyacyl CoA dehydrogenase (SCHAD) is a soluble dimeric enzyme critical for oxidative metabolism of fatty acids. Its primary sequence has been reported to be conserved across numerous tissues and species with the notable exception of the pig heart homologue. Preliminary efforts to solve the crystal structure of the dimeric pig heart SCHAD suggested the unprecedented occurrence of three enzyme subunits within the asymmetric unit, a phenomenon that was thought to have hampered refinement of the initial chain tracing. The recently solved crystal coordinates of human heart SCHAD facilitated a molecular replacement solution to the pig heart SCHAD data. Refinement of the model, in conjunction with the nucleotide sequence for pig heart SCHAD determined in this paper, has demonstrated that the previously published pig heart SCHAD sequence was incorrect. Presented here are the corrected amino acid sequence and the high resolution crystal structure determined for pig heart SCHAD complexed with its NAD+ cofactor (2.8 A; R(cryst) = 22.4%, R(free) = 28.8%). In addition, the peculiar phenomenon of a dimeric enzyme crystallizing with three subunits contained in the asymmetric unit is described.

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