Hydroxylation of 4-hydroxyphenylethylamine derivatives by R263 variants of the oxygenase component of p-hydroxyphenylacetate-3-hydroxylase.
Arch Biochem Biophys
; 620: 1-11, 2017 04 15.
Article
em En
| MEDLINE
| ID: mdl-28300536
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.
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MEDLINE
Assunto principal:
Proteínas de Bactérias
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Tiramina
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Mutação de Sentido Incorreto
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Acinetobacter baumannii
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Oxigenases de Função Mista
Idioma:
En
Ano de publicação:
2017
Tipo de documento:
Article