Microbial transformations of steroids—V. Transformation of progesterone by whole cells and extracts of Botryosphaerica obtusa

Abstract

Members of the genus Botryosphaerica are reported 7α steroid hydroxylators [1], We found that the species B. obtusa efficiently hydroxylated progesterone in a 1-day transformation but it gave 7β-hydroxyprogesterone as the main product rather than the expected 7β-hydroxy isomer, which was produced in only trace amounts. Also formed in minor amounts were 6β-, possibly 9α-(see main text), 14α- and 15β-monohydroxyprogesterones. The transformation mixtures included appreciable amounts of dihydroxylated progesterones which were mainly based on 7β-hydroxyprogesterone. The second hydroxyl group was at one of the minor monohydroxylation sites. The relative concentrations of the progesterone diols increased and those of the mono-alcohols concomitantly decreased when transformation was extended beyond 1 day. Monohydroxylated 6-dehydroprogesterones began to accumulate after about 3 days and these compounds seemed to have been formed by 6,7-dehydration of the dihydroxyprogesterones. We prepared mycelial cell-free extracts which were capable of transforming progesterone and retained the site-specificity of whole cells. These extracts converted 7β-hydroxyprogesterone to its 6-dehydro derivative, confirming that ring B desaturation occurs in this organism by dehydration. The dehydratase activity necessary for the conversion was separable from the hydroxylase activity by ultra-centrifugation. All hydroxylase activity co-sedimented with the membrane fraction, implying that steroid hydroxylation is effected by a membrane-bound enzyme(s). Dehydratase activity was present in both the pellet and the supernatant fractions, which suggests that it may involve a loosely bound, and easily removed, membrane-associated enzyme.