title: Hydroxylation of 4-hydroxyphenylethylamine derivatives by R263 variants of the oxygenase component of p-hydroxyphenylacetate-3-hydroxylase.
abstract: p-hydroxyphenylacetate 3-hydroxylase from Acinetobacter baumannii catalyzes the hydroxylation of p-hydroxyphenylacetate (HPA) to yield 3,4-dihydroxyphenylacetate (DHPA). In this study, we investigated whether variants of the oxygenase component (C2) could catalyze hydroxylation of 4-hydroxyphenylethylamines to synthesize catecholamine derivatives. Single turnover product analysis showed that the R263D variant can catalyze hydroxylation of tyramine to form dopamine with the highest yield (57%). The enzyme was also found to have dual substrate charge specificity because it can also maintain reasonable hydroxylation efficiency of HPA (86%). This property is different from the R263E variant, which can hydroxylate HPA (73%) but not tyramine. The R263A variant can hydroxylate HPA (72%) and tyramine to a small extent (7%). Stopped-flow experiments indicated that tyramine and HPA prefer binding to R263D after C4a-hydroperoxy-FMN formation, while tyramine cannot bind to the wild-type or R263E enzymes. Data also indicate that the hydroxylation rate constant is the rate-limiting step. The R263D variant was used as a starting enzyme for further mutation to obtain other variants for the synthesis of additional catecholamine drugs. The R263D/Y398D double mutant enzyme showed interesting results in that it was able to catalyze the hydroxylation of octopamine to form norepinephrine. However, the enzyme still lacked stereo-selectivity in its reaction.
authors.affiliation: Institute for Innovative Learning, Mahidol University, Nakhon Pathom 73170, Thailand. Electronic address: pirom.che@mahidol.ac.th.
authors.forename: Pirom
authors.initials: P
authors.lastname: Chenprakhon
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 Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand.
authors.forename: Rattikan
authors.initials: R
authors.lastname: Chantiwas
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: D040981:Acinetobacter baumannii / Q000201:enzymology* / Q000235:genetics; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D006900:Hydroxylation; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics; D020125:Mutation, Missense; D009638:Norepinephrine / Q000737:chemistry; D009655:Octopamine / Q000737:chemistry; D014439:Tyramine / Q000031:analogs & derivatives* / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D009655:Octopamine; C085265:3-methoxy-4-hydroxyphenylethylamine; D006899:Mixed Function Oxygenases; C026252:4-hydroxyphenylacetate 3-monooxygenase; D009638:Norepinephrine; D014439:Tyramine
keywords: 4-hydroxyphenylethylamine; Catecholamine; Dopamine; Flavin-dependent two-component monooxygenase; Tyramine; p-hydroxyphenylacetate 3-hydroxylase