Arunachalam U, Massey V, Vaidyanathan C S
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor 48109-0606.
J Biol Chem. 1992 Dec 25;267(36):25848-55.
p-Hydroxyphenylacetate-3-hydroxylase, an inducible enzyme isolated from the soil bacterium Pseudomonas putida, catalyzes the conversion of p-hydroxyphenylacetate to 3,4-dihydroxyphenylacetate. The enzyme requires two protein components: a flavoprotein and a colorless protein referred to as the coupling protein. The flavoprotein alone in the presence of p-hydroxyphenylacetate and substrate analogs catalyzes the wasteful oxidation of NADH with the stoichiometric generation of H2O2. A 1:1 complex of the flavoprotein and coupling protein is required for stoichiometric product formation. Such complex formation also eliminates the nonproductive NADH oxidase activity of the flavoprotein. A new assay measuring the product formation activity of the enzyme was developed using homoprotocatechuate-2,3-dioxygenase, as monitoring the oxidation of NADH was not sufficient to demonstrate enzyme activity. The coupling protein does not seem to have any redox center in it. Thus, this 2-component flavin hydroxylase resembles the other aromatic hydroxylases in that the only redox chromophore present is FAD.
对羟基苯乙酸-3-羟化酶是一种从土壤细菌恶臭假单胞菌中分离得到的可诱导酶，它催化对羟基苯乙酸转化为3,4-二羟基苯乙酸。该酶需要两种蛋白质组分：一种黄素蛋白和一种被称为偶联蛋白的无色蛋白。单独的黄素蛋白在对羟基苯乙酸和底物类似物存在的情况下，催化NADH的无效氧化，并按化学计量生成过氧化氢。为了按化学计量生成产物，黄素蛋白和偶联蛋白需要形成1:1的复合物。这种复合物的形成也消除了黄素蛋白的非生产性NADH氧化酶活性。利用原儿茶酸-2,3-双加氧酶开发了一种新的测定该酶产物形成活性的方法，因为监测NADH的氧化不足以证明酶的活性。偶联蛋白似乎不含有任何氧化还原中心。因此，这种双组分黄素羟化酶与其他芳香族羟化酶相似，因为唯一存在的氧化还原发色团是FAD。
