Mucor hiemalis, a filamentous fungus, transforms progesterone primarily through 14α-hydroxylation, producing 14α-hydroxyprogesterone and subsequently multiple dihydroxylated derivatives. A novel metabolite, 8(9),14(15)-didehydroprogesterone, was identified. Ketoconazole inhibition confirmed that the hydroxylase enzymes are cytochrome P-450 monooxygenases. Unexpectedly, transcription inhibitors (actinomycin D) and heat-stress stimulated progesterone hydroxylation, while the translation inhibitor cycloheximide blocked activity throughout treatment but paradoxically activated the enzyme when applied only during pre-incubation. These contrasting effects suggest that progesterone 14α-hydroxylase mRNA is normally sequestered in an inactive form by a labile regulatory protein. Upon blocking transcription or heat-stress, the sequestering protein's synthesis ceases, releasing stored mRNA for translation. This regulation model explains substrate-induced enzyme activation and contrasts with standard induction mechanisms observed in other cytochrome P-450 systems.

Key findings

• Mucor hiemalis predominantly hydroxylates progesterone at the 14α position via cytochrome P-450 enzymes, producing a novel metabolite 8(9),14(15)-didehydroprogesterone not previously reported in microbial systems
• Transcription inhibitors and heat-stress stimulate progesterone hydroxylation while translation inhibitors prevent it, opposite to typical P-450 gene regulation patterns
• The data support an mRNA sequestration model where cytochrome P-450 hydroxylase mRNA is stored in dormant form bound to a labile sequestering protein that dissociates upon transcription block or stress
• Substrate pre-incubation with progesterone or 14α-hydroxyprogesterone increases enzyme activity, suggesting induction of the hydroxylases
• Ketoconazole inhibition confirms the hydroxylases are cytochrome P-450 monooxygenases dependent on NADPH electron transport