title: Identification of a gatekeeper residue that prevents dehydrogenases from acting as oxidases.
abstract: The oxygen reactivity of flavoproteins is poorly understood. Here we show that a single Ala to Gly substitution in l-galactono-gamma-lactone dehydrogenase (GALDH) turns the enzyme into a catalytically competent oxidase. GALDH is an aldonolactone oxidoreductase with a vanillyl-alcohol oxidase (VAO) fold. We found that nearly all oxidases in the VAO family contain either a Gly or a Pro at a structurally conserved position near the C4a locus of the isoalloxazine moiety of the flavin, whereas dehydrogenases prefer another residue at this position. Mutation of the corresponding residue in GALDH (Ala-113 --> Gly) resulted in a striking 400-fold increase in oxygen reactivity, whereas the cytochrome c reductase activity is retained. The activity of the A113G variant shows a linear dependence on oxygen concentration (k(ox) = 3.5 x 10(5) m(-1) s(-1)), similar to most other flavoprotein oxidases. The Ala-113 --> Gly replacement does not change the reduction potential of the flavin but creates space for molecular oxygen to react with the reduced flavin. In the wild-type enzyme, Ala-113 acts as a gatekeeper, preventing oxygen from accessing the isoalloxazine nucleus. The presence of such an oxygen access gate seems to be a key factor for the prevention of oxidase activity within the VAO family and is absent in members that act as oxidases.
authors.affiliation: Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands.
authors.forename: Nicole G H
authors.initials: NG
authors.lastname: Leferink
authors.forename: Marco W
authors.initials: MW
authors.lastname: Fraaije
authors.forename: Henk-Jan
authors.initials: HJ
authors.lastname: Joosten
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Schaap
authors.forename: Andrea
authors.initials: A
authors.lastname: Mattevi
authors.forename: Willem J H
authors.initials: WJ
authors.lastname: van Berkel
mesh_terms: D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics; D019943:Amino Acid Substitution; D000596:Amino Acids / Q000737:chemistry / Q000235:genetics; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics; D005420:Flavoproteins / Q000737:chemistry* / Q000235:genetics; D020125:Mutation, Missense; D010084:Oxidation-Reduction; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000737:chemistry* / Q000235:genetics; D010100:Oxygen / Q000737:chemistry
chemical_list: D000596:Amino Acids; D029681:Arabidopsis Proteins; D005420:Flavoproteins; D000429:Alcohol Oxidoreductases; C076865:vanillyl-alcohol oxidase; D044925:Oxidoreductases Acting on CH-CH Group Donors; C093910:galactonolactone dehydrogenase; D010100:Oxygen