Glutamate 170 of Human l-3-Hydroxyacyl-CoA Dehydrogenase Is Required for Proper Orientation of the Catalytic Histidine and Structural Integrity of the Enzyme

Abstract

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...

Crystal Structures of Transhydrogenase Domain I with and without Bound NADH,

“…In structures of enzymes containing NADH or NADPH, the dihydronicotinamide ring stacks parallel with substrate or flavin, and the C4 atom is within 3.0-3.5 Å of the hydride acceptor, consistent with direct hydride transfer (27)(28)(29)(30). Except for glutathione reductase (30), these enzymes follow the correlation that transfer from the pro-R (A) face of the dihydronicotinamide is favored when the glycosyl torsion angle is anti while transfer from the pro-S (B) face is favored when it is syn (23).…”

Identification by Mutagenesis of Conserved Arginine and Glutamate Residues in the C-terminal Domain of Rat Liver Carnitine Palmitoyltransferase I That Are Important for Catalytic Activity and Malonyl-CoA Sensitivity

“…Furthermore, the site-directed mutagenesis studies demonstrate that even a conservative substitution of Glu-603 to aspartate or glutamine resulted in partial loss of activity and malonyl-CoA sensitivity, suggesting that a change of Glu-603 to aspartate may result in the carboxylate being outside the hydrogen bond distance of His-473. Glu-603 may thus be required for L-CPTI stability and positioning of the imidazole ring of His-473 for efficient catalysis and inhibition, thus facilitating productive interaction with the substrates and the inhibitor (23). Mutation of the corresponding conserved residue in CPTII, Glu-500, to alanine resulted in 50% loss in activity (24).…”

“…As a rate-limiting enzyme that transports long chain fatty acids from the cytosol to the mitochondrial matrix, L-CPTI in the presence of carnitine catalyzes the conversion of long chain acyl CoAs to acylcarnitines (1,2). Similar to other acyltransferases, L-CPTI contains a general acid/base, His-473, a highly conserved amino acid residue that may form a hydrogen bonding network or a salt bridge to a nearby conserved glutamate residue such as Glu-603 (23). We hypothesize that substitution of Glu-603 with alanine or histidine may disrupt a hydrogen bonding network or a salt bridge, perhaps to the highly conserved residue His-473 that is predicted to be at the active site of L-CPTI.…”

The Multifunctional Protein in Peroxisomal β-Oxidation

“…The 3-hydroxyacyl-CoA dehydrogenase signature is indicated by the magenta color of the backbone trace. The proposed reaction mechanism of AtMFP2 is based on the present crystal structure and the reaction mechanism and structures of human HACD NAD ϩ and its substrate analogue complexes (56,70).…”

“…Also included is the superimposed acetyl-CoA from the same PfMFP structure. The conserved residues His 449 and Glu 461 have been identified as being important for dehydrogenase activity by sitedirected mutagenesis of E. coli MFP, and a reaction mechanism has been proposed and later modified (56,69,70). The reaction mechanism implies that the hydrogen bond between Glu 461 and His 449 would orientate His 449 for optimal proton subtraction from the substrates hydroxyl group.…”