3‐Ketosteroid‐9α‐Hydroxylase

Abstract

KshAB is a Rieske oxygenase (RO) in the aerobic steroid degradation pathways of bacteria. It is a monooxygenase responsible for the 9α‐hydroxylation of the nucleus of 3‐keto‐4‐ene steroids bearing short side chains at C17. It is composed of two domains arranged in a head‐to‐tail fashion to form the functional trimer. The N‐terminal Rieske domain harbors a Fe 2 S 2 cluster in which the two irons are coordinated by two cysteine and two histidine residues, respectively. The larger, C‐terminal catalytic domain contains a mononuclear nonheme iron coordinated by a His‐His‐Asp facial triad. Purified KshAB from Mycobacterium tuberculosis exhibits substrate specificities for 1,4‐androstadien‐3,17‐one and dioxygen 2‐3 orders of magnitude lower than those measured for other ROs and their best substrates. In agreement with the large fused‐ring substrate, the structure of KshA incorporates an active site channel and substrate binding pocket that are longer than those observed in other ROs, and positioned in a different relative orientation within the catalytic domain. KshA also possesses a C‐terminal helix which occupies a position not observed in other RO structures and which likely stabilizes trimeric quaternary structure. Finally, KshA exhibits a minimal core catalytic domain with respect to secondary structural elements that may prove archetypical for ROs.