The Steroid-9α-Hydroxylation System from Nocardia Species

Abstract

The steroid 9u-hydroxylase from Nocardia species M117 was found to be an electron-transport chain consisting of an NADH-dependent flavoprotein reductase and two iron-sulfur proteins named protein I1 and protein 111. The components were partially purified. The flavoprotein reductase from Nocardia species M117 was enriched 20-fold to 100 units/mg and protein 111 200-fold to 2400 units/mg protein. Protein I1 has a molecular weight of approximately 214000. The purification factor of protein I1 was not determined. The absorption spectrum of protein I1 shows a maximum at 425 nm in the oxidized form and maxima at 510 nm, 415 nm and 370 nm in the reduced state; whereas protein 111 has a prominent maximum at 452 nm in the oxidized form and no absorption maximum in the reduced state. Carbon monoxide treatment of the reduced forms of protein I1 and protein 111 showed no maximum at 450 nm. Both proteins I1 and 111 are sensitive to oxygen.The hydroxylase activity can be reconstituted from the isolated components. Activity of the combined proteins was demonstrated by product analysis and NADH consumption produced by the addition of progesterone. Protein 111 catalyzes the reduction of cytochrome c in the presence of NADH and Nocardia flavoprotein reductase.The reconstitution of hydroxylase activity from cytochrome-free enzyme preparations excludes the participation of cytochrome P-450 in this steroid hydroxylase system. Stereospecific hydroxylation by microorganisms is a wellused tool in research and industry [I -51 for the introduction of oxygen at defined non-activated positions of the molecules. Nonetheless, knowledge about microbial hydroxylases is limited.It is difficult to follow activity during enzyme purification procedures because hydroxylases work by a concerted action of different proteins according to an electron-transport chain, no simple assays are available for the individual links of the system and the protein components are rather unstable in vitro (61.Some hydroxylase systems which have been resolved into their components contain cytochrome P-450. Among the steroid-hydroxylating systems the 15-fi-hydroxylase from Bacillus megaterium was shown to contain cytochrome P-450 [7]. One of the most thoroughly investigated hydroxylase system, the camphor 5-exo-monooxygenases from Pseudomonas putida, uses cytochrome P-450 as terminal oxidase [8,8a]. In recent years many different cytochrome P-450-containing hydroxylases have been discovered. But this should not lead to the assumption that this kind of terminal oxidase is the most widespread species of hydroxylase systems. There are some hydroxylases described in which iron-sulfur proteins act as terminal oxidases [9-12al. The number of well-investigated species of this kind of hydroxylase is low, perhaps due to the difficulties in purification and characterization of this protein class.The steroid 9a-hydroxylase, which is a key-enzyme in the steroid-ring-B-splitting pathway, occurs in many bacterial genera used in industrial processes [I 31, e.g. Nocardia, Mycobacteriu...

Effect of Tween 80 on 9α-steroid hydroxylating activity and ultrastructural characteristics of Rhodococcus sp. cells

“…In this connection it is necessary to recall that the function of 9a-steroid hydroxylase is coupled with a wellorganized membrane-bond electron-transport chain (Strijewski 1982). Recent investigations proved that ferredoxin reductase, a terminal oxygenase and NADH are essential for the 9a-hydroxylase activity with steroid substrates (Petrusma et al 2009).…”

Characterization of 3-Ketosteroid 9α-Hydroxylase, a Rieske Oxygenase in the Cholesterol Degradation Pathway of Mycobacterium tuberculosis

“…In addition, the oxygenase component was structurally characterized. Previous attempts to purify a ketosteroid hydroxylase were hampered by the O 2 lability of one or more of the components (56). Although the terminal oxygenase of that enzyme, which was also from an actinomycete, was not identified, the enzyme appeared to be a three-component system.…”

Degradation of and sensitivity to cholate in Pseudomonas sp. strain Chol1

“…Anaerobic degradation was limited to the formation of DHADD because its further degradation to THSATD depended on an oxygenasedependent reaction step which we detected in the soluble fraction of the cell-free extract. Such 9a-steroidhydroxylases have been found in Gram-positive bacteria (Strijewski 1982;van der Geize et al 2002). Altogether, aerobic cholate degradation by strain Chol1 appears to proceed through the route that Hayakawa (1982) proposed for Gram-negative bacteria.…”