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, 16alpha-hydroxyprogesterone was produced in 3.6 fold excess to the minor metabolite 2beta,16alpha-dihydroxyprogesterone. In a reconstituted system containing highly purified progesterone 16alpha-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 0.32 mM progesterone and the purified components of the hydroxylase system incubated as before, the ratio of 16alpha-hydroxyprogesterone to 2beta,16alpha-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 16alpha-hydroxyprogesterone to 2beta,16alpha-dihydroxyprogesterone ratio was also obtained when S. roseochromogenes 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.
Berrie
J R
JR
Molecular and Cellular Biology, Division of Biomedical Sciences, Queen Mary and Westfield College Medical School, University of London, Mile End Road, London E1 4NS, UK.
Williams
R A
RA
Smith
K E
KE
D001426:Bacterial Proteins / Q000378:metabolism*; D002474:Cell-Free System; D003513:Cycloheximide / Q000494:pharmacology; D004579:Electron Transport; D004591:Electrophoresis, Polyacrylamide Gel; D006900:Hydroxylation; D007525:Isoelectric Focusing; D011374:Progesterone / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D039181:Steroid 16-alpha-Hydroxylase; D013256:Steroids / Q000378:metabolism*; D013302:Streptomyces / Q000378:metabolism*
D001426:Bacterial Proteins; D013256:Steroids; D011374:Progesterone; D003513:Cycloheximide; D039181:Steroid 16-alpha-Hydroxylase