Rhodococcus rhodochrous DSM 43269 3-ketosteroid 9alpha-hydroxylase, a two-component iron-sulfur-containing monooxygenase with subtle steroid substrate specificity.

ABSTRACT: This paper reports the biochemical characterization of a purified and reconstituted two-component 3-ketosteroid 9alpha-hydroxylase (KSH). KSH of Rhodococcus rhodochrous DSM 43269, consisting of a ferredoxin reductase (KshB) and a terminal oxygenase (KshA), was heterologously expressed in Escherichia coli. E. coli cell cultures, expressing both KshA and KshB, converted 4-androstene-3,17-dione (AD) into 9alpha-hydroxy-4-AD (9OHAD) with a >60% molar yield over 48 h of incubation. Coexpression and copurification were critical to successfully obtain pure and active KSH. Biochemical analysis revealed that the flavoprotein KshB is an NADH-dependent reductase using flavin adenine dinucleotide as a cofactor. Reconstitution experiments confirmed that KshA, KshB, and NADH are essential for KSH activi

SUBMITTER: Petrusma M 

PROVIDER: S-EPMC2725467 | biostudies-literature | 2009 Aug

REPOSITORIES: biostudies-literature