Search

The introduction of a 16 alpha-hydroxyl function into the steroid nucleus was studied in resting cells of Streptomyces roseochromogenes NRRL B-1233. The oxidation product of dehydroepiandrosterone (DHEA) was identified as 16 alpha-hydroxy DHEA by using thin-layer and gas-liquid chromatography. A linear relation between cell concentration and 16 alpha-OH-DHEA formation was observed. 16 alpha-Hydroxylase showed good activity at pH 8.0 for 16 alpha-OH-DHEA formation. The enzyme showed good activity at 3.1 x 10(-4) M DHEA. The oxidation products of pregnenolone, 4-androstene-3,17-dione, estrone, and 5-androstene-3 beta,17 beta-diol as well as of other substrates were identified as the 16 alpha-hydroxy steroid, respectively. The rates of microbial 16 alpha-hydroxylation were as follows: 76.9% for DHEA, 50.4% for pregnenolone, 43.9% for 4-androstene-3,17-dione, 34.3% for estrone, and 19.6% for 5-androstene-3 beta,17 beta-diol. The organism tested catalyzes 16 alpha-hydroxylation of a wide variety of steroids.

[14-14C]16 alpha-Hydroxy-C-18- and C-19-steroid hormones were obtained in good yields by microbiological hydroxylation of correspondingly labelled steroids by Streptomyces roseochromogenes NRRL B-1233. Trace quantities of the labelled substrates were incubated on a rotary shaker (220 rpm) at 27 degrees C. The radioactive products were chromatographically separated, identified and the radiochemical purity was established by isotopic dilution analysis. The specific activities of 16 alpha-hydroxy-steroids obtained were assumed to be the same as those of the substrates, namely, 57.5 mCi/mmole for 16 alpha-hydroxy-4-androstene-3,17-dione, 57.5 mCi/mmole for 5-androstene-3 beta,16 alpha,17 beta-triol, 57.5 mCi/mmole for 16 alpha-hydroxy-dehydroepiandrosterone, 55.7 mCi/mmole for 16 alpha-hydroxy-estrone, and 57.5 mCi/mmole for 16 alpha-hydroxy-testosterone.

Streptomyces roseochromogenes (NRRL B-1233) converted estrone predominantly into its 16 alpha-hydroxyl derivative. Chemical and spectroscopic (UV, IR, NMR, MS) methods were used in establishing the structure and strereochemistry of the oxidation product. The product was assigned as 16 alpha-hydroxyestrone (yield, 17%). No other oxidation product was detected in this experiment. An interrelationship between cell growth and 16 alpha-hydroxy-estrone formation was observed. Also, 16 alpha-hydroxylation of estrone was observed in resting cells. 16 alpha-Hydroxylase showed good activity at 3.7 X 10(-4)M of estrone concentration and was completely inhibited by 1.1 X 10(-3)M. These results indicate the presence of a constitutive 16 alpha-hydroxylase in the organism investigated.

The microbial transformation of a sesquiterpene lactone, 1-alpha-santonin was carried out by Streptomyces roseochromogenes NRRL-B-1233. The product obtained from 1-alpha-santonin was purified using silica gel column chromatography with 1% acetone-benzene as the eluant. The molecular weight of the product was 248, estimated by mass spectroanalysis. The product was assigned the chemical structure of 1,2-dihydro-1-alpha santonin on the bases of 1H and 13C-NMR and mass spectroanalyses. Similar products were also obtained from other actinomycetes. The conversion of 1-alpha-santonin to 1,2-dihydro-1-alpha-santonin was 28% for Streptomyces roseochromogenes NRRL-B-1233, 14% for S. roseochromogenes ATCC 13400, 30.9% for S. aureofaciens KCC-S-0008, and 43.0% for S. aureofaciens KCC-S-0624, respectively.

Biosynthesis of [7-3H]16alpha-hydroxy-dehydroepiandrosterone in high specific activity has been studied. [7-3H] dehydroepiandrosterone (13.9 C/mM) in trace quantity was oxidized by Streptomyces roseochromogenes (NRRLB-1233) for 5 min at 27 degrees C. The radioactive products were chromatographically separated, identified and their radiochemical purity established by isotopic dilution analysis. [7-3H]16alpha-hydroxy-dehydroepiandrosterone (2.5 x 10(7) dpm) was obtained by microbial hydroxylation of substrate (1.9 X 10(9) dpm). In some cases [7-3H])5-androstene-3beta, 16alpha, 17beta-triol in a small amount of radioactivity could be found at the prolonged reaction for 30 hr.