title: A Single-Site Mutation at Ser146 Expands the Reactivity of the Oxygenase Component of p-Hydroxyphenylacetate 3-Hydroxylase.
abstract: The oxygenase component (C2) of p-hydroxyphenylacetate (4-HPA) 3-hydroxylase (HPAH) from Acinetobacter baumannii catalyzes the hydroxylation of various phenolic acids. In this report, we found that substitution of a residue close to the phenolic group binding site to yield the S146A variant resulted in an enzyme that is more effective than the wild-type in catalyzing the hydroxylation of 4-aminophenylacetate (4-APA). Product yields for both wild-type and S146A enzymes are better at lower pH values. Multiple turnover reactions of the wild-type and S146A enzymes indicate that both enzymes first hydroxylate 3-APA to give 3-hydroxy-4-aminophenylacetate (3-OH-4-APA), which is further hydroxylated to give 3,5-dihydroxy-4-aminophenylacetate, similar to the reaction of C2with 4-HPA. Stopped-flow experiments showed that 4-APA can only bind to the wild-type enzyme at pH 6.0 and not at pH 9.0, while it can bind to S146A under both pH conditions. Rapid-quench flow results indicate that the wild-type enzyme has low reactivity toward 4-APA hydroxylation, with a hydroxylation rate constant (kOH) for 4-APA of 0.028 s-1compared to 17 s-1for 4-HPA, the native substrate. In contrast, for S146A, the hydroxylation rate constants for both substrates are very similar (2.6 s-1for 4-HPA versus 2.5 s-1for 4-APA). These data indicate that Ser146 is a key catalytic residue involved in optimizing C2reactivity toward a phenolic compound. Removing this hydroxyl group expands C2activity toward a non-natural aniline substrate. This understanding should be helpful for future rational engineering of other two-component flavin-dependent monooxygenases that have this conserved Ser residue.
authors.affiliation: Department of Biochemistry and Center for Excellence in Protein and Enzyme Technology, Faculty of Science, Mahidol University , Rama 6 Road, Bangkok 10400, Thailand.
authors.forename: Taweesak
authors.initials: T
authors.lastname: Dhammaraj
authors.affiliation: Department of Biochemistry and Center for Excellence in Protein and Enzyme Technology, Faculty of Science, Mahidol University , Rama 6 Road, Bangkok 10400, Thailand.|Institute for Innovative Learning, Mahidol University , Nakhon Pathom 73170, Thailand.
authors.forename: Chatchadaporn
authors.initials: C
authors.lastname: Pinthong
authors.affiliation: Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Mahidol University , Rama 6 Road, Bangkok 10400, Thailand.
authors.forename: Surawit
authors.initials: S
authors.lastname: Visitsatthawong
authors.affiliation: Department of Biochemistry and Center for Excellence in Protein and Enzyme Technology, Faculty of Science, Mahidol University , Rama 6 Road, Bangkok 10400, Thailand.
authors.forename: Chanakan
authors.initials: C
authors.lastname: Tongsook
authors.affiliation: Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Mahidol University , Rama 6 Road, Bangkok 10400, Thailand.
authors.forename: Panida
authors.initials: P
authors.lastname: Surawatanawong
authors.affiliation: Department of Biochemistry and Center for Excellence in Protein and Enzyme Technology, Faculty of Science, Mahidol University , Rama 6 Road, Bangkok 10400, Thailand.
authors.forename: Pimchai
authors.initials: P
authors.lastname: Chaiyen
mesh_terms: D002384:Catalysis; D006863:Hydrogen-Ion Concentration; D006900:Hydroxylation; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D012694:Serine / Q000235:genetics*; D013053:Spectrophotometry / Q000379:methods; D013379:Substrate Specificity
chemical_list: D012694:Serine; D006899:Mixed Function Oxygenases