title: Biochemical characterization of human 3-methylglutaconyl-CoA hydratase and its role in leucine metabolism.
abstract: The metabolic disease 3-methylglutaconic aciduria type I (MGA1) is characterized by an abnormal organic acid profile in which there is excessive urinary excretion of 3-methylglutaconic acid, 3-methylglutaric acid and 3-hydroxyisovaleric acid. Affected individuals display variable clinical manifestations ranging from mildly delayed speech development to severe psychomotor retardation with neurological handicap. MGA1 is caused by reduced or absent 3-methylglutaconyl-coenzyme A (3-MG-CoA) hydratase activity within the leucine degradation pathway. The human AUH gene has been reported to encode for a bifunctional enzyme with both RNA-binding and enoyl-CoA-hydratase activity. In addition, it was shown that mutations in the AUH gene are linked to MGA1. Here we present kinetic data of the purified gene product of AUH using different CoA-substrates. The best substrates were (E)-3-MG-CoA (V(max) = 3.9 U.mg(-1), K(m) = 8.3 microM, k(cat) = 5.1 s(-1)) and (E)-glutaconyl-CoA (V(max) = 1.1 U.mg(-1), K(m) = 2.4 microM, k(cat) = 1.4 s(-1)) giving strong evidence that the AUH gene encodes for the major human 3-MG-CoA hydratase in leucine degradation. Based on these results, a new assay for AUH activity in fibroblast homogenates was developed. The only missense mutation found in MGA1 phenotypes, c.719C>T, leading to the amino acid exchange A240V, produces an enzyme with only 9% of the wild-type 3-MG-CoA hydratase activity.
authors.affiliation: Institut für Technische Mikrobiologie der Hochschule Mannheim, Germany. m.mack@hs-mannheim.de
authors.forename: Matthias
authors.initials: M
authors.lastname: Mack
authors.forename: Ute
authors.initials: U
authors.lastname: Schniegler-Mattox
authors.forename: Verena
authors.initials: V
authors.lastname: Peters
authors.forename: Georg F
authors.initials: GF
authors.lastname: Hoffmann
authors.forename: Michael
authors.initials: M
authors.lastname: Liesert
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Buckel
authors.forename: Johannes
authors.initials: J
authors.lastname: Zschocke
mesh_terms: D000214:Acyl Coenzyme A / Q000378:metabolism; D000592:Amino Acid Metabolism, Inborn Errors / Q000175:diagnosis / Q000201:enzymology / Q000235:genetics; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005347:Fibroblasts / Q000201:enzymology; D005977:Glutarates / Q000378:metabolism; D006801:Humans; D006836:Hydro-Lyases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D007700:Kinetics; D007930:Leucine / Q000378:metabolism*; D009154:Mutation; D013379:Substrate Specificity
chemical_list: D000214:Acyl Coenzyme A; D005977:Glutarates; C022034:3-methylglutaconic acid; C032423:glutaconyl-coenzyme A; D006836:Hydro-Lyases; C035867:methylglutaconyl-CoA hydratase; D007930:Leucine