Cloning of glutaryl-CoA dehydrogenase cDNA, and expression of wild type and mutant enzymes in <i>Escherichia coli</i>

Goodman, Stephen I.; Kratz, Lisa E.; DiGiulio, Kathleen Axtell; Biery, Barbara J.; Goodman, Karen E.; Isaya, Grazia; Frerman, Frank E.

doi:10.1093/hmg/4.9.1493

Cited by 69 publications

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“…Previous structural and enzymatic studies of GCDH have repeatedly demonstrated FAD to be a tightly bound cofactor for this enzyme. Human GCDH recombinantly expressed and purified in another laboratory by methods similar to those used here revealed FAD bound in the crystal structure, although FAD had not been explicitly added at any stage (Dwyer et al, 2000;Goodman et al, 1995;Fu et al, 2004). Biochemical efforts to remove FAD from Pseudomonas fluorescens GCDH in another laboratory required multiple acid-ammonium precipitation steps, generating a very unstable protein which lost all activity if FAD was not added back within minutes (Gomes et al, 1981).…”

Section: Discussionmentioning

confidence: 88%

“…Glutaryl-CoA dehydrogenase (GCDH) is an acyl-CoA dehydrogenase (ACDH) which catalyzes an intermediate step in the metabolic breakdown of lysine and tryptophan (Gomes et al, 1981;Kim & Miura, 2004;Lenich & Goodman, 1986;Goodman et al, 1995). Unlike other flavoproteins within this family, GCDH (EC 1.3.99.7) performs , -dehydrogenation as well as decarboxylation of its substrate glutaryl-CoA to yield the product crotonyl-CoA (Dwyer et al, 2000).…”

Section: Introductionmentioning

confidence: 99%

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Probing conformational states of glutaryl-CoA dehydrogenase by fragment screening

Begley

Davies

Hartley

et al. 2011

Acta Crystallogr F Struct Biol Cryst Commun

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Glutaric acidemia type 1 is an inherited metabolic disorder which can cause macrocephaly, muscular rigidity, spastic paralysis and other progressive movement disorders in humans. The defects in glutaryl-CoA dehydrogenase (GCDH) associated with this disease are thought to increase holoenzyme instability and reduce cofactor binding. Here, the first structural analysis of a GCDH enzyme in the absence of the cofactor flavin adenine dinucleotide (FAD) is reported. The apo structure of GCDH from Burkholderia pseudomallei reveals a loss of secondary structure and increased disorder in the FAD-binding pocket relative to the ternary complex of the highly homologous human GCDH. After conducting a fragment-based screen, four small molecules were identified which bind to GCDH from B. pseudomallei. Complex structures were determined for these fragments, which cause backbone and side-chain perturbations to key active-site residues. Structural insights from this investigation highlight differences from apo GCDH and the utility of small-molecular fragments as chemical probes for capturing alternative conformational states of preformed protein crystals.

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Section: Discussionmentioning

confidence: 88%

Section: Introductionmentioning

confidence: 99%

Probing conformational states of glutaryl-CoA dehydrogenase by fragment screening

Begley

Davies

Hartley

et al. 2011

Acta Crystallogr F Struct Biol Cryst Commun

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“…Some of these dehydrogenases, e.g. medium chain acyl-CoA dehydrogenase (MCAD), are involved in ␤-oxidation of fatty acids (19), whereas others, including glutaryl-CoA dehydrogenase (GCDH) and isovalerylCoA dehydrogenase, are involved in the oxidation of amino acids (20,21). Yet another group is involved in oxidative reactions in the pathway for degradation of choline to glycine, resulting in the transfer of one-carbon moieties.…”

mentioning

confidence: 99%

Human METTL20 Is a Mitochondrial Lysine Methyltransferase That Targets the β Subunit of Electron Transfer Flavoprotein (ETFβ) and Modulates Its Activity

Małecki

Moen

et al. 2015

Journal of Biological Chemistry

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Background: Many proteins are modified by lysine methylation. Results: It is shown that the previously uncharacterized enzyme METTL20 methylates electron transfer flavoprotein ␤ (ETF␤), thereby inhibiting its ability to mediate electron transfer from acyl-CoA dehydrogenases. Conclusion: METTL20-mediated methylation modulates the function of ETF␤. Significance: The first mitochondrial lysine-specific protein methyltransferase in animals is reported, and the resulting methylation is shown to have functional consequences.

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“…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).…”

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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Cloning of glutaryl-CoA dehydrogenase cDNA, and expression of wild type and mutant enzymes in Escherichia coli

Cited by 69 publications

References 0 publications

Probing conformational states of glutaryl-CoA dehydrogenase by fragment screening

Probing conformational states of glutaryl-CoA dehydrogenase by fragment screening

Human METTL20 Is a Mitochondrial Lysine Methyltransferase That Targets the β Subunit of Electron Transfer Flavoprotein (ETFβ) and Modulates Its Activity

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

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