Microbial transformation of steroids—VII. hydroxylation of progesterone by extracts of Phycomyces blakesleeanus

Abstract

Post mitochondrial supernatants (S-12 extracts) were prepared from Phycomyces blakesleeanus by grinding washed and frozen mycelial cakes in fine sand and extracting the paste produced with buffer containing Tris-HCl pH 7.8 (0.1 M), EDTA (0.01 M), dithiothreitol (5 mM) and glycerol (10% v/v). The S-12 extracts, obtained in this way, reproducibly hydroxylated progesterone, producing 7α- and 15β-hydroxyprogesterone the major products of whole-cell transformation. Cell-free progesterone hydroxylation was found to be approximately linearly dependent on extract concentration, to require reduced NADP (partly replaceable by NADH), and to be dependent on progesterone (apparent K m calculated to be 4 mM). K + and Mg 2+ were found not to be required. Maximum progesterone hydroxylation occurred after 2 h at pH 7.8 and at 24°C. Using optimum conditions S-12 extracts were capable of hydroxylating between 5 and 15% of added progesterone (0.2 mM). Hydroxylation was found to be partially inhibited by carbon monoxide ( ca 40%) and almost completely inhibited by azoles, ketoconazole and diconazole. The NADPH and molecular oxygen requirements were replaceable by NaIO 4. These findings strongly suggest that hydroxylation was being catalyzed by cytochrome P-450. This was confirmed by preparing progesterone-hydroxylating microsomes and Triton N-101-solubilized microsome extracts, and by obtaining a dithionite-reduced carbon monoxide-difference absorption spectrum peak at 455 nm in the solubilized microsome extracts.