Hydroxylation of 4-hydroxyphenylethylamine derivatives by R263 variants of the oxygenase component of p-hydroxyphenylacetate-3-hydroxylase

作者：Chenprakhon Pirom^*; Dhammaraj Taweesak; Chantiwas Rattikan; Chaiyen Pimchai

来源：Archives of Biochemistry and Biophysics, 2017, 620: 1-11.

DOI：10.1016/j.abb.2017.03.004

• Flavin-dependent two-component monooxygenase
• p-hydroxyphenylacetate 3-hydroxylase
• 4-hydroxyphenylethylamine
• Catecholamine
• Tyramine
• Dopamine

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 (C-2) 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.

• 出版日期2017-4-15