K. Schubert

Steroid transforming enzymes from microorganisms IV. Purification and cofactor requirement of the 4-ene-3-oxosteroid-5α-reductase from Mycobacterium smegmatis

4-Ene-3-Oxosteroid-5α-reductase of Mycobacterium smegmatis is bound with the subcellular structure of the microbial cell, although about 50% of activity can be released by sonication. By using some procedures of protein fractionation—separation of nucleic acids, salt precipitation, gel filtration and ion exchange chromatography—the enzyme was purified about 60-fold with a recovery of 36%. 5α-Reductase activity was not stimulated by NADH or NADPH. The FAD-dependent character of the enzyme was deduced from its activation by dithionite, inhibition by acriflavin and restoration of activity after inactivation by acidic ammonium sulphate. The conception of a possible role of the flavin is discussed.

Abbau Von Steroiden: XIII. Hydroxylierung von 17α-methylöstradiol in 2-, 4- und 6α-stellung durch Aspergillus flavus

Abstract 2-, 4- and 6α-Monohydroxylation products of 17α-methylestradiol ∗ have been obtained from fermentation cultures of Aspergiltus fiavus. The physical properties and the kinetical investigation of the microbiological metabolites are discussed.

Steroid transforming enzymes from microorganisms—III: Properties of 4-ene-3-oxosteroid-5α-reductase from Mycobacterium smegmatis

Abstract Sonicated cell-free extract of M . smegmatis is capable of reducing 4-ene-3-oxosteroids to the corresponding 5α-dihydro-products. In the presence of sodium dithionite and with 4-androstenedione as substrate 5α-reduetion takes place as the predominant transformation reaction. The enzyme remains in the 105,000 g supernatant and can be precipitated by ammonium sulfate at 30–45% saturation. The enzyme can be stored at — 20° for at least 3 months without inactivation. At room temperature it loses about 50% activity in 48 h. In phosphate buffer the pH optimum is at 6.3 and the temperature optimum at 40°. Molecular weight is about 40,000. Thiol reagents, heavy metal ions and o -phenanthroline are strong inhibitors of the 5α-reductase, The inhibition by acriflavin and the increasing effect of sodium dithionite indicates the participation of flavin-nucleotides. Progesterone, testosterone and derivatives with additional double bonds and substituents in positions 4 and 17α, as well as both d - and l -19-nortestosterone are transformed to 5α-dihydrocompounds. The apparent k m for androstenedione is 7.5 × 10 −6 M. Cyproterone and Chlormadinonacetate are powerful steroid inhibitors of the enzyme.

Abbau von steroiden: XII. Enzymatische bildung eines 5-ring-ketons aus dem entsprechenden δ-lakton beim abbau von progesteron durch den pilz Aspergillus flavus

Abstract Aspergillus flavus degradates progesterone in the same way as bacteria, but an alternative pathway leads through testololactone to a corresponding keto acid with the remaining lactone ring. At this stage of metabolism the lactone ring was enzymatically reconverted into the 5-ring ketone.

Abbau von steroiden—XIV. Bildung von [1-14C]-bernsteinsäure und [5-14C]-lävulinsäure aus 7a-methyl-5,6,7,7a-tetrahydroind an-[5-14C]-1,5-dion-4-(3-propionsäure-[1-14C]) durch Nocardia opaca

Abstract The mechanism of microbial degradation of steroid rings B and C was investigated. The intermediate 7a-methyl-5,6,7,7a-tetrahydroindan-l,5-dione-4-(3-propionic acid) was synthesized, 14C labelled in position 1 of propionic acid (position 5 of sterane skeleton) and position 5 of tetrahydroindane (position 9 of sterane skeleton) respectively. From the labelled intermediate [1-14C]-succinic acid and [1-14C]-levulinic acid could be obtained by fermentation with Nocardia opaca. Besides this the same intermediate has been 14C-labelled in the methyl-group of position 7α (position 18 of sterane skeleton). From this labelled intermediate only [5-14C]-levulinic acid was obtained. The formation of these specific labelled acids could be seen in accordance with the further degradation of the 5-methyl-4-oxooctane-1,8-dioic-acid isolated from Hayakawa[1], With these findings the microbial degradation of the sterane skeleton is elucidated in all positions.

Mikrobielle hydrierung des aromatischen systems von 17α-äthinylöstradiol

Abstract The first enzymatic hydrogenation of an aromatic system of steroids is described. 17α-Ethinylestradiol (17α-Ethinyl-l,3,5(10)-estratriene-3,17β-diol) has been transformed by Aspergillus flavus to 10β-Hydroxyl-19-nor-ethisterone (17α-Ethinyl-10β,17β-dithydroxy-4-estren-3-one).

Abbau von Steroiden:—XV. Bildung von 9,10-seco-verbindungen aus 4-chlorsubstituierten steroiden durch Nocardia opaca

Zusammenfassung 4-Chlortestosterone (V), the corresponding 17α-methylsubstituted steroid (II) and the compound with an additional double bond in position 1 (I) have been transformed to 9,10-Seco-compounds (IV and VII) by 9α-hydroxylation and 1-dehydrogenation in fermentation cultures of Nocardia opaca . Compound I has been hydrogenated in position 1 to compound II. The metabolisation of compound V could be markedly increased by addition of estrone.