Pirom ChenprakhonTaweesak DhammarajRattikan ChantiwasPimchai ChaiyenMahidol University2018-12-212019-03-142018-12-212019-03-142017-04-15Archives of Biochemistry and Biophysics. Vol.620, (2017), 1-1110960384000398612-s2.0-85015815753https://repository.li.mahidol.ac.th/handle/20.500.14594/41927© 2017 Elsevier Inc. 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.Mahidol UniversityBiochemistry, Genetics and Molecular BiologyArticleSCOPUS10.1016/j.abb.2017.03.004