Identification of the Active Site Catalytic Residue in Human Isovaleryl-CoA Dehydrogenase

Mohsen, Al-Walid A.; Vockley, Jerry

doi:10.1021/bi00032a007

Cited by 48 publications

(58 citation statements)

References 15 publications

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“…Glu261 is topologically conserved in the three-dimensional structure but lies in a different segment of the polypeptide chain in the primary sequences (Figure 1). A similar study (Mohsen & Vockley, 1995) confirmed that the catalytic base in IVDH is Glu254 (255 in MCADH numbering). In the preceding paper, a converse experiment has been performed in which both Glu376 and Thr255 of MCADH have been altered to the corresponding residues found in LCADH, glycine, and glutamate, respectively.…”

mentioning

confidence: 54%

Crystal Structures of the Wild Type and the Glu376Gly/Thr255Glu Mutant of Human Medium-Chain Acyl-CoA Dehydrogenase: Influence of the Location of the Catalytic Base on Substrate Specificity

et al. 1996

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Crystal structures of the wild type human medium-chain acyl-CoA dehydrogenase (MCADH) and a double mutant in which its active center base-arrangement has been altered to that of long chain acyl-CoA dehydrogenase (LCADH), Glu376Gly/Thr255Glu, have been determined by X-ray crystallography at 2.75 and 2.4 Å resolution, respectively. The catalytic base responsible for the R-proton abstraction from the thioester substrate is Glu376 in MCADH, while that in LCADH is Glu255 (MCADH numbering), located over 100 residues away in its primary amino acid sequence. The structures of the mutant complexed with C8-, C12, and C14-CoA have also been determined. The human enzyme structure is essentially the same as that of the pig enzyme. The structure of the mutant is unchanged upon ligand binding except for the conformations of a few side chains in the active site cavity. The substrate with chain length longer than C12 binds to the enzyme in multiple conformations at its ω-end. Glu255 has two conformations, "active" and "resting" forms, with the latter apparently stabilized by forming a hydrogen bond with Glu99. Both the direction in which Glu255 approaches the C R atom of the substrate and the distance between the Glu255 carboxylate and the C R atom are different from those of Glu376; these factors are responsible for the intrinsic differences in the kinetic properties as well as the substrate specificity. Solvent accessible space at the "midsection" of the active site cavity, where the C R -C bond of the thioester substrate and the isoalloxazine ring of the FAD are located, is larger in the mutant than in the wild type enzyme, implying greater O 2 accessibility in the mutant which might account for the higher oxygen reactivity.Acyl-CoA dehydrogenases are a family of flavoenzymes that catalyze the R, -dehydrogenation of acyl-CoA thioesters to the corresponding trans-enoyl-CoA with transfer of reducing equivalents to electron transfer flavoprotein (Beinert, 1963). In mammalian mitochondria, four straight-chain specific acyl-CoA dehydrogenases [short (SCADH)-, medium (MCADH)-, long (LCADH)-, and very long chain (VLCAD) acyl-CoA dehydrogenase] involved in fatty acid catabolism (Beinert, 1963;Aoyama et al., 1994) and three branched-chain specific acyl-CoA dehydrogenases (isovaleryl-, 2-methylbutyryl-, and glutaryl-CoA-DH) in amino acid metabolism have been described (Tanaka & Indo, 1992;Lenich & Goodman, 1986). Among them, MCADH is the most studied: the three dimensional structure of pig MCADH has been determined with and without various substrates and inhibitors (Kim et al., 1993 and its catalytic residue has been identified (Powell & Thorpe, 1988) and confirmed by site-specific mutagenesis (Bross, et al, 1990) and by the three dimensional structures of enzyme-substrate complex (Kim et al., 1993). All seven acyl-CoA dehydrogenases have been cloned, sequenced, and over-expressed in bacterial systems. Of the 27 known amino acid sequences of acylCoA dehydrogenases from both mammalian and bacterial sources available, the catal...

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confidence: 54%

Crystal Structures of the Wild Type and the Glu376Gly/Thr255Glu Mutant of Human Medium-Chain Acyl-CoA Dehydrogenase: Influence of the Location of the Catalytic Base on Substrate Specificity

et al. 1996

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“…1) and transfers the reducing equivalents to the electron transfer flavoprotein [Ikeda et al, 1983;Ikeda and Tanaka, 1988;Mohsen and Vockley 1995]. It is encoded in the nuclear genome as a precursor protein, but is active in the mitochondrial matrix [Ikeda et al, 1987;Parimoo and Tanaka, 1993;Vockley et al, 1991].…”

Section: Molecular Findingsmentioning

confidence: 99%

Isovaleric acidemia: New aspects of genetic and phenotypic heterogeneity

Vockley

Ensenauer

2006

American J of Med Genetics Pt C

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192

161

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Isovaleric acidemia (IVA) is an autosomal recessive inborn error of leucine metabolism caused by a deficiency of the mitochondrial enzyme isovaleryl-CoA dehydrogenase (IVD) resulting in the accumulation of derivatives of isovaleryl-CoA. It was the first organic acidemia recognized in humans and can cause significant morbidity and mortality. Early diagnosis and treatment with a protein restricted diet and supplementation with carnitine and glycine are effective in promoting normal development in severely affected individuals. Both intra-and inter-familial variability have been recognized. Initially, two phenotypes with either an acute neonatal or a chronic intermittent presentation were described. More recently, a third group of individuals with mild biochemical abnormalities who can be asymptomatic have been identified through newborn screening of blood spots by tandem mass spectrometry. IVD is a flavoenzyme that catalyzes the conversion of isovaleryl-CoA to 3-methylcrotonyl-CoA and transfers electrons to the electron transfer flavoprotein. Human IVD has been purified from tissue and recombinant sources and its biochemical and physical properties have been extensively studied. Molecular analysis of the IVD gene from patients with IVA has allowed characterization of different types of mutations in this gene. One missense mutation, 932C>T (A282V), is particularly common in patients identified through newborn screening with mild metabolite elevations and who have remained asymptomatic to date. This mutation leads to a partially active enzyme with altered catalytic properties; however, its effects on clinical outcome and the necessity of therapy are still unknown. A better understanding of the heterogeneity of this disease and the relevance of genotype/phenotype correlations to clinical management of patients are among the challenges remaining in the study of this disorder in the coming years.

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“…Anaerobic Titration of Wild Type and Mutant SBCADs with Substrate-Wild type and mutant enzymes were titrated with (S)-2-methylbutyryl-CoA or hexanoyl-CoA as described previously (20,30,35) with minor modifications. Spectral scans were performed using a Beckman DU7400 spectrophotometer (Palo Alto, CA) equipped with a computer.…”

Section: Construction Of Sbcad Prokaryotic Expression Vectors-mentioning

confidence: 99%

A Novel Approach to the Characterization of Substrate Specificity in Short/Branched Chain Acyl-CoA Dehydrogenase

Burghardt

Vockley

2003

Journal of Biological Chemistry

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Rat and human short/branched chain acyl-CoA dehydrogenases exhibit key differences in substrate specificity despite an overall amino acid identity of 85% between them. Rat short/branched chain acyl-CoA dehydrogenases (SBCAD) are more active toward substrates with longer carbon side chains than human SBCAD, whereas the human enzyme utilizes substrates with longer primary carbon chains. The mechanism underlying this difference in substrate specificity was investigated with a novel surface plasmon resonance assay combined with absorbance and circular dichroism spectroscopy, and kinetics analysis of wild type SBCADs and mutants with altered amino acid residues in the substrate binding pocket. Results show that a relatively few amino acid residues are critical for determining the difference in substrate specificity seen between the human and rat enzymes and that alteration of these residues influences different portions of the enzyme mechanism. Molecular modeling of the SBCAD structure suggests that position 104 at the bottom of the substrate binding pocket is important in determining the length of the primary carbon chain that can be accommodated. Conformational changes caused by alteration of residues at positions 105 and 177 directly affect the rate of electron transfer in the dehydrogenation reactions, and are likely transmitted from the bottom of the substrate binding pocket to ␤-sheet 3. Differences between the rat and human enzyme at positions 383, 222, and 220 alter substrate specificity without affecting substrate binding. Modeling predicts that these residues combine to determine the distance between the flavin ring of FAD and the catalytic base, without changing the opening of the substrate binding pocket.The acyl-CoA dehydrogenases (ACDs) 1 are a family of related enzymes that catalyze the ␣, ␤-dehydrogenation of acylCoA esters, transferring electrons to electron transferring flavoprotein (1-4). Deficiencies of these enzymes are important causes of human disease (3-7). Biochemical and immunological studies have identified at least 9 distinct members of this enzyme family, each having a characteristic substrate utilization pattern (8 -13). Very long, long, medium, and short chain acyl-CoA dehydrogenases (VLCAD, LCAD, MCAD, and SCAD, respectively) catalyze the first step in the mitochondrial ␤-oxidation of fatty acids with substrate optima of 16, 16, 8, and 4 carbon chains, respectively (3, 14 -15). A new ACD has been identified recently (ACAD9) that is also active against long chain acyl-CoA substrates (16). Isovaleryl-CoA dehydrogenase (IVD), short/branched chain acyl-CoA dehydrogenase (SBCAD, also known as 2-methyl branched chain acyl-CoA dehydrogenase), and isobutyryl-CoA dehydrogenase (IBD) catalyze the third step in leucine, isoleucine, and valine metabolism, respectively (2-4, 11, 12, 17, 18), whereas glutaryl-CoA dehydrogenase is active in lysine metabolism (19).We have shown previously that SCAD, SBCAD, and IBD can all utilize butyryl-CoA as substrate (albeit with different efficiencies), whereas ...

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Identification of the Active Site Catalytic Residue in Human Isovaleryl-CoA Dehydrogenase

Cited by 48 publications

References 15 publications

Crystal Structures of the Wild Type and the Glu376Gly/Thr255Glu Mutant of Human Medium-Chain Acyl-CoA Dehydrogenase: Influence of the Location of the Catalytic Base on Substrate Specificity

Crystal Structures of the Wild Type and the Glu376Gly/Thr255Glu Mutant of Human Medium-Chain Acyl-CoA Dehydrogenase: Influence of the Location of the Catalytic Base on Substrate Specificity

Isovaleric acidemia: New aspects of genetic and phenotypic heterogeneity

A Novel Approach to the Characterization of Substrate Specificity in Short/Branched Chain Acyl-CoA Dehydrogenase

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