Microbial transformations of steroids-XI.: Progesterone transformation by Streptomyces roseochromogenes-purification and characterisation of the 16α-hydroxylase system

Streptomyces roseochromogenes, NCIB 10984, contains a cytochrome P450 which, in conjunction with two indigenous electron transfer proteins, roseoredoxin and roseoredoxin reductase, hydroxylates exogenous progesterone firstly to 16 alpha-hydroxyprogesterone and thereafter in a second phase bioconversion to 2 beta, 16 alpha-dihydroxyprogesterone. The progesterone 16 alpha-hydroxylase P450 and the two electron transfer proteins have been purified to homogeneity. A reconstituted incubation containing these three purified proteins a nd NADH, the natural electron do nor, produced identical hydroxy-progesterone metabolites as in intact cells. Peroxy and hydroperoxy compounds act in a shortened form of the cycle known as the 'peroxide shunt' by replacing the natural pathway requirement for the electron donor NADH, the electron transfer proteins and molecular O-2, the terminal electron acceptor. In an NaIO4 supported incubation, the initial rate of progesterone hydroxylation was marginally higher (1.62 mmol progesterone/mmol P-450/h) than in the reconstituted natural incubation (1.18 mmol progesterone/mmol P-450/h) but the product yield was significantly lower, 0.45 mol hydroxyprogesterone produced/mol P-450 compared to 6.0 mol hydroxyprogesterone produced/mol P-450. These yield data show that in the reconstituted natural pathway, progesterone 16 alpha-hydroxylase P450 supports multiple rounds of hydroxylation in contrast to a likely single oxygenation by a minority of P450s in the peroxide shunt pathway. (C) 2000 Elsevier Science Ltd. All rights reserved.