By I. A. EL-KADY
Department of Botany, Faculty of Science, Assiut University, Assiut, Egypt
(Received 28 August 1981 ; revised 19 January 1982)
Growing cultures of Aspergillus niger hydroxylate progesterone at the 6P and 1 la positions. The properties of the 6p-hydroxylase activity were studied in extracts of this organism using llahydroxyprogesterone as substrate. Maximum 6P-hydroxylase activity was obtained at pH 6.5. EDTA and thiophenol inhibited hydroxylation, while NADPH, 2-oxoglutarate and pyruvate stimulated it. Cobalt and cadmium ions inhibited the 6P-hydroxylase activity. The feasibility of introducing a hydroxyl group into the 6P-position of various steroid compounds was tested : the 3-0X0-A4 configuration appeared to exert a directional effect for 6p-hydroxylation, while an 118-
hydroxyl group exerted some kind of steric hindrance against 6p-hydroxylation.
The important discovery by Peterson & Murray (1952) of the microbiological 1 la-hydroxylation of progesterone and other steroids led to the use of a variety of micro-organisms for hdyroxylating every position in the steroid nucleus. However, steroid hydroxylation by extracts of micro-organisms has been described in only a few instances. Zuidweg et al. (1962) prepared an extract from Curvularia Zunata capable of catalysing the hydroxylation of Reichstein’s compound S to hydrocortisone. A 9a-hydroxylase activity was demonstrated in extracts of Nocardia restrictus by Chang & Sih (1964). Wilson & Vestling (1965) obtained an extract from Bacillus megaterium KM able to hydroxylate deoxycorticosterone mainly at the 15p position. Zuidweg (1968), using extracts of C. Zunata, studied the nature of the hydroxylation of Reichstein’s compound S at the 1lP and 14a positions. Sallam et al. (1971), using a cell homogenate of Rhizopus nigricans REF 129, studied the hydroxylation of progesterone at the 1 la, 17a and 21 positions. Nguyen-Dang-Tam et al. (1971) showed that extracts of Aspergillus niger catalysed the hydroxylation of progesterone at the 1 la position. Previously, I have studied the hydroxylation of progesterone at the 1 la and 17a positions by extracts of Rhizopusnigricans NRRL 1477 (El-Kady & Allam, 1973; Allam & El-Kady, 1975). In this report, I describe an investigation of the 6P-hydroxylation of progesterone and 1 la-hydroxyprogesterone by extracts of Aspergillus niger.
Cultures. Aspergillus niger isolate no. 58 (from the Culture Collection of the Mycological Laboratory, Botany Department, Assiut University, Egypt) was cultivated in 250 ml Erlenmeyer flasks which contained 50 ml of the medium described by Capek et al. (1964). The pH was adjusted to 5.6. After sterilization, each flask was inoculated with 2ml of a spore suspension made from a 1-week old culture. The inoculated flasks were agitated on a reciprocating shaker at 28 “C. After 24 h growth, the cultures were activated by the addition of 5 mg progesterone per flask and reagitated for another 24 h.
Preparation of extracts. Mycelium was harvested by filtration, washed, thoroughly with 0.5 % (w/v) NaCl solution, followed by distilled water, and blotted dry with absorbent paper. This mycelium (1 5 g) was then ground with cold sand and extracted with 100 ml cold water or 0.1 M-pOtaSSiUm phosphate buffer, pH 6-5. The slurry obtained was centrifuged at 4000 rev. min-‘ for 15 min and the supernatant was used as the enzyme source.
Assay of6P-hydroxylating activity in extracts. To 5 ml portions of the extracts in 25 ml Erlenmeyer flasks were added 5 mg 1 la-hydroxyprogesterone in 0-1 ml ethanol. The contents were mixed immediately and the flasks were agitated on a reciprocating shaker for 5 h at 30°C. At the end of this period the contents of each flask were extracted with 10 ml chloroform. Extraction was repeated three times and the combined chloroform extracts were then treated with 0.5 vol. of 5% (w/v) sodium bicarbonate solution followed by an equal volume of distilled water. The residue was dissolved in a measured volume of chloroform/methanol (1 : 1, by vol.). Determination of reaction products. The hydroxylated product in the prepared residue was first identified by conventional thin-layer chromatography with an authentic sample, using cyclohexane/acetone/chloroform (75 : 25 : 20, by vol.) as solvent. The amounts of the product and residual substrate were determined by preparative thin-layer chromatography with the same solvent. The bands containing the compounds were scraped off and eluted with ethanol. The concentration of each was then determined by U.V. spectrophotometric measurement.
RESULTS AND DISCUSSION
Out of 40 different isolates from five genera (Rhizopus, Mucor, Cunninghamella, Cladosporium and Aspergillus) tested for 61-hydroxylation activity, Aspergillus niger isolate no. 58 was the most active and was chosen for further study. Growing cultures of this organism converted 80% of added progesterone (50 mg per 50 ml medium) to 1 la-hydroxyprogesterone (10 mg) and 6p,1 ladihydroxyprogesterone (30 mg) within 48 h. Using 1 la-hydroxyprogesterone as a substrate, the stability of the 6P-hydroxylating activity was tested in extracts stored at 5 and – 10 “C. Extracts kept at 5 “C lost 90% of their activity in 24 h and those kept at – 10 “C lost 60% of their activity in the same period. After 48 h at either temperature all activity was lost. In earlier studies (Chang & Sih, 1964; El-Kady & Allam, 1973) microbial steroid-hydroxylating systems were shown to be unstable.
Efect of pH. The effect of different pH values on the 6P-hydroxylating activity was studied. Portions (4 ml) of the extract in distilled water were adjusted to pH values ranging from 5-5 to 9 using phosphate or Tris buffers (500 pmol). Hydroxylation at the 6P position increased with increase in pH, reaching a maximum at pH 6-5. At this pH, 42% of the added 1 la-hydroxyprogesterone was converted to 6p, 1 la-dihydroxyprogesterone. At pH values between 7 and 9, the 6P-hydroxylase activity decreased linearly with pH.
Efect of EDTA. EDTA has been used in most studies of the hydroxylation of steroids by extracts of micro-organisms (Zuidweg et al., 1962; Chang & Sih, 1964; Zuidweg, 1968; Sallam et al., 1971 ; El-Kady & Allam, 1973). The effect of different concentrations of this compound on the 6P-hydroxylating activity was tested. The presence of EDTA at a concentration as low as 5 mM inhibited about 30% of the 6P-hydroxylase activity. Higher concentrations of EDTA were more inhibitory (data not presented). This suggests that a metal cation is involved in the hydroxylation.
Eflect of some external electron carriers and thiol compounds. A marked stimulation of 6phydroxylase activity was observed in the presence of NADPH (Table 1). 2-Oxoglutarate and pyruvate had a similar effect, but ascorbate, fumarate and succinate were completely inactive. These results are consistent with earlier reports that 2-oxoglutarate stimulates different hydroxylation reactions (Hayaishi, 1969), and that pyruvate can replace 2-oxoglutarate in some enzymic hydroxylation reactions (Hayaishi, 1969).
The effect of some thiol compounds was also studied (Table 2). Glutathione, at a concentration of 5 mM, stimulated 6P-hydroxylase activity, while 2-mercaptoethanol, sodium thioglycolate and cysteine.HC1 had no effect. On the other hand, thiophenol, at the same concentration, caused some inhibition.
Efect of some metal ions. The 6P-hydroxylase activity was sensitive to Co2+ and Cd2+ which inhibited 80% and 60% of the 6P-hydroxylase activity, respectively (Table 3). Previous reports (El-Kady & Allam, 1973) showed that Co2+ completely inhibited both 1 la- and 17a-hydroxylase activities.
Substrate specijicity. The feasibility of introducing a hydroxyl group into the 68 position of some steroid compounds was tested. The following compounds were equally active as substrates for the 6P-hydroxylase : progesterone, 1 1 a-hydroxyprogesterone, 17a-hydroxyprogesterone, 21 – hydroxyprogesterone, 1 1 a, 17a-dihydroxyprogesterone, epicortisol, testosterone, 1 1 a hydroxytestosterone and androst-4-ene-3,17-dione. In contrast, the following compounds were found to be inactive for the 6p-hydroxylase : 1 1 p-hydroxyprogesterone, 1 1 or-hydroxy-5a-pregnane-3,20-dione, 17a-hydroxy-5a-pregnane-3,20-dione, 5a-pregnane-3p, 17a,21-triol-2O-one, 1 1/3,21-dihydroxyprogesterone, cortisol, cholesterol and ergosterol. It was noted that all the active substrates contained the 3-oxo-A4 configuration. Hence, this configuration probably exerts a directional effect for 6Q-hydroxylation. The inactive compounds lack at least one of these two functional groups (3-oxo-A4-), except 1 1 p-hydroxyprogesterone, 1 1p,2 1 -dihydroxyprogesterone and cortisol which contain the 3-oxo-A4 configuration but also possess an 1 1 P-hydroxyl group. Thus, it appears that an 1 lp-hydroxyl group exerts some kind of’ steric hindrance against 6phydroxylat ion.
Table 1. Efect of some electron carriers on 6P-hydroxylation
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