Microbial transformations of steroids-XII.: Progesterone hydroxylation profiles are modulated by post-translational modification of an electron transfer protein in Streptomyces roseochromogenes

When Streptomyces roseochromogenes strain 10984 was incubated with exogenous progesterone for 25 h the major monohydroxylated metabolite, 16 alpha -hydroxyprogesterone was produced in 3.6 fold excess to the minor metabolite 2 beta ,16 alpha -dihydroxyprogesterone. In a reconstituted system containing highly purified progesterone 16 alpha -hydroxylase cytochrome P-450, and electron transfer proteins ferredoxin-like redoxin (roseoredoxin) and redoxin reductase (roseoredoxin reductase), both metabolites were produced but in a 10:1 ratio. When S. roseochromogenes was pre-incubated for 8 h with 0.33 mM progesterone and the purified components of the hydroxylase system incubated as before, the ratio of 16 alpha -hydroxyprogesterone to 2 beta ,16 alpha -dihydroxyprogesterone produced decreased to 2.8:1, virtually identical to the ratio in whole cell transformations. Reconstitution assays containing all combinations of hydroxylase proteins purified from progesterone pre-incubated and control cells showed that the roseoredoxin was solely responsible for the observed changes in in vitro metabolite ratios. The fact that the lower 16 alpha -hydroxyprogesterone to 2 beta ,16 alpha -dihydroxyprogesterone ratio was also obtained when S. roseochuomogenes was exposed to 0.335 mM cycloheximide for 8 h prior to the progesterone pre-incubation, pointed to post-translation modification of the roseoredoxin. Separation of two isoforms of roseoredoxin by isoelectric focusing supported this proposition. (C) 2001 Elsevier Science Ltd. All rights reserved.