p-Hydroxyphenylacetate-3-hydroxylase. A two-protein component enzyme.
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.
Arunachalam
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U
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor 48109-0606.
Massey
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Vaidyanathan
C S
CS
D000693:Anaerobiosis; D002852:Chromatography, Ion Exchange; D049308:Dioxygenases; D004591:Electrophoresis, Polyacrylamide Gel; D005420:Flavoproteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D010105:Oxygenases / Q000302:isolation & purification* / Q000378:metabolism; D016958:Pseudomonas putida / Q000201:enzymology* / Q000254:growth & development; D013053:Spectrophotometry; D013379:Substrate Specificity
D005420:Flavoproteins; D009243:NAD; D006899:Mixed Function Oxygenases; D010105:Oxygenases; D049308:Dioxygenases; C020055:3,4-dihydroxyphenylacetate 2,3-dioxygenase; C026252:4-hydroxyphenylacetate 3-monooxygenase