Heterogeneous natures of the microbial steroid 9alpha-hydroxylase in nocardioforms

Steroid 9alpha-hydroxylase is an enzyme found in nocardioform microorganisms which can utilize steroids as a sole carbon source. After fractional centrifugation of the cell homogenates, the enzyme activity inNocardia andRhodococcus was found in cytoplasmic membrane fraction. On the contrary,Mycobacterium had its 9alpha-hydroxylation activity in cytosolic fraction. To characterize the enzyme in these microorganisms, several potential inhibitors of 9alpha-hydroxylase were tested and the cofactor requirement for the same enzyme was also examined. The inhibitory effect of ferrous ion chelators indicated involvement of iron containing proteins in the 9alpha-hydroxylase system. On the other hand, metyrapone, an inhibitor known to be specific for cytochrome P450 interfered with the enzyme inMycobacterium, but didn't inhibit the enzyme activity inNocardia andRhodococcus. While the 9alpha-hydroxylase system inNocardia andRhodococcus required NADPH, NADH was required as an election donor inMycobacterium.