Abstract:
Indole, a bicyclic compound comprising a fused benzene and pyrrole ring, serves as a fundamental component in numerous natural compounds. Additionally, indole holds significant importance in industrial applications, particularly in the production of pharmaceutical drugs and agrochemicals. Flavin-dependent indole monooxygenase is an enzyme responsible for hydroxylation of the indole ring. Indole monooxygenase is a two-components: flavoprotein reductase containing FAD as a cofactor, and monooxygenase. The reductase component utilizes NADH to catalyze flavin reduction and transfers the reduced flavin to the monooxygenase component. The hydroxylation of indole by monooxygenase reduced FAD and oxygen as substrates. In this study, we cloned the indole monooxygenase (IndOx) from the genome of Acinetobacter baumannii and the gene was subcloned into an expression vector with a C-terminal histidine tag. The expression IndOx resulted in a high yield of 0.31 g/L, while reductase (IndR) yield of 0.11 g/L in the culture media. To elucidate the enzyme mechanism, rapid kinetic experiments were conducted using a stopped-flow spectrophotometer. The hydroxylation of indole was found a specific hydroxylation at the C3 position of indole ring, resulting in the production of 3-hydroxyindole. Understanding the enzyme reaction mechanism will be applied for enzyme engineering for production of indigoid compounds for diverse applications in medicines, cosmetics, food industries, and the manufacturing of high-value materials.
