A novel two-protein component flavoprotein hydroxylase.
p-Hydroxyphenylacetate (HPA) hydroxylase (HPAH) was purified from Acinetobacter baumannii and shown to be a two-protein component enzyme. The small component (C1) is the reductase enzyme with a subunit molecular mass of 32 kDa. C1 alone catalyses HPA-stimulated NADH oxidation without hydroxylation of HPA. C1 is a flavoprotein with FMN as a native cofactor but can also bind to FAD. The large component (C2) is the hydroxylase component that hydroxylates HPA in the presence of C1. C2 is a tetrameric enzyme with a subunit molecular mass of 50 kDa and apparently contains no redox centre. FMN, FAD, or riboflavin could be used as coenzymes for hydroxylase activity with FMN showing the highest activity. Our data demonstrated that C2 alone was capable of utilizing reduced FMN to form the product 3,4-dihydroxyphenylacetate. Mixing reduced flavin with C2 also resulted in the formation of a flavin intermediate that resembled a C(4a)-substituted flavin species indicating that the reaction mechanism of the enzyme proceeded via C(4a)-substituted flavin intermediates. Based on the available evidence, we conclude that the reaction mechanism of HPAH from A. baumannii is similar to that of bacterial luciferase. The enzyme uses a luciferase-like mechanism and reduced flavin (FMNH2, FADH2, or reduced riboflavin) to catalyse the hydroxylation of aromatic compounds, which are usually catalysed by FAD-associated aromatic hydroxylases.
Chaiyen
P
P
Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, Thailand. scpcy@mahidol.ac.th
Suadee
C
C
Wilairat
P
P
D000150:Acinetobacter / Q000201:enzymology*; D005486:Flavin Mononucleotide / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005415:Flavins / Q000378:metabolism; D006900:Hydroxylation; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
C071365:3-hydroxyphenylacetate 6-hydroxylase; D005415:Flavins; D009097:Multienzyme Complexes; D005182:Flavin-Adenine Dinucleotide; D005486:Flavin Mononucleotide; D006899:Mixed Function Oxygenases