title: Purification and characterization of a Galactomyces reessii hydratase that converts 3-methylcrotonic acid to 3-hydroxy-3-methylbutyric acid.
abstract: Cell free extracts of Galactomyces reessii contain a hydratase as the key enzyme for the transformation of 3-methylcrotonic acid to 3-hydroxy-3-methylbutyric acid. Highest levels of hydratase activity were obtained during growth on isovaleric acid. The enzyme, an enoyl CoA hydratase, was purified 147-fold by precipitation with ammonium sulphate and successive chromatography over columns of DE-52, Blue Sepharose CL-6B and Sephacryl S-200. During purification, hydratase activity was measured spectrophotometrically (OD change at 263 nm) for 3-methylcrotonyl CoA and crotonyl CoA as substrates. The enzyme displayed highest activity with crotonyl CoA with a Kcat of 1,050,000 min(-1). The ratio of crotonyl CoA to 3-methylcrotonyl CoA activities was constant (20:1) during all steps of purification. The Kcat for crotonyl CoA was also about 20 times greater than the Kcat for 3-methylcrotonyl CoA (51,700 min(-1). The enzyme had pH and temperature optima at 7.0 and 35 degrees C, a native Mr of 260 +/- 4.5 kDa and a subunit Mr of 65 kDa, suggesting that the enzyme was a homotetramer. The pI of the purified hydratase was 5.5, and the N-terminal amino acid sequence was VPEGYAEDLLKGKMMRFFDS. Hydratase activity for 3-methylcrotonyl CoA was competitively inhibited by acetyl CoA, propionyl CoA and acetoacetyl CoA.
authors.affiliation: Division of Medicinal and Natural Products Chemistry, Center For Biocatalysis and Bioprocessing, College of Pharmacy, University of Iowa, Iowa City 52242, USA.
authors.forename: A
authors.initials: A
authors.lastname: Dhar
authors.forename: K
authors.initials: K
authors.lastname: Dhar
authors.forename: J P N
authors.initials: JP
authors.lastname: Rosazza
mesh_terms: D000595:Amino Acid Sequence; D002087:Butyrates / Q000378:metabolism*; D002384:Catalysis; D004746:Enoyl-CoA Hydratase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D004790:Enzyme Induction; D008969:Molecular Sequence Data; D004718:Saccharomycetales / Q000378:metabolism*
chemical_list: D002087:Butyrates; D004746:Enoyl-CoA Hydratase