Distribution of steroid 1-dehydrogenation and side-chain degradation enzymes in the spores of Fusarium solani: causes of metabolic lag and carbohydrate independence.

The spores of a strain of Fusarium solani 1-dehydrogenate ring A and cleave the 17beta-acetyl side chain of 17alpha-hydroxypregn-4-ene-3,20-dione (17alpha-hydroxyprogesterone) to give 17alpha-hydroxypregna-1,4-diene-3,20-dione (the 1-dehydro analogue) and little androsta-1,4-diene-3,4-diene-3,17-dione (androstadienedione). A 4-h lag period is observed in the course of metabolism, and there are no requirements for external additives. Exoenzymes or surface enzymes bound to the cell outside the plasma membrane, either in the periplasmic space or bound to the cell wall, cannot be detected. The spore activity is not destroyed by treatment with aqueous HCl (pH 1.50), indicating that the 1-dehydrogenation and side-chain degradation enzymes are located away from the surface of the spores. Phenethyl alcohol destroys the spore permeability barriers, and it is also likely that it exposes its enzymes to acid inactivation. The action of phenethyl alcohol is reversible at low concentrations and irreversible at high concentrations. This investigation shows that: (i) the spore 1-dehydrogenating and side-chain-degrading enzymes appear to be bound to, or imbedded in, the plasma membrane; (ii) the lag period observed in the course of metabolism of the steroid by the spores might be required for enzyme activation or diffusion of the substrate through the cell wall; and (iii) the internal metabolities of the spores, that might be required for the conversion process, appear to be present in a nondiffusible form or bound to intrasporal macromolecules.

Reduction of the 20-carbonyl group of C-21 steroids by spores of Fusarium solani and other microorganisms. I. Side-chain degradation, epoxide cleavage, and substrate specificity.

The spores of Fusarium solani reduced the C(2)-carbonyl group, 1-dehydrogenated ring "A" and cleaved the side chain of 16alpha, 17alpha-oxidopregn-4-ene-3, 20-dione (16alpha, 17alpha-oxidoprogesterone)(I) to give the following products: 20alpha-hydroxy-16alpha, 17alpha-oxidopregn-4-en-3-one(II); 20alpha-hydroxy-16alpha, 17alpha-oxidopregna-1, 4-dien-3-one(III); 16alpha-hydroxy-17a-oxa-androsta-1, 4-diene-3, 17-dione (16alpha-hydroxy-1-dehydrotestololactone)(IV); and 16alpha, 17beta-dihydroxy-androsta-1, 4-dien-3-one (16alpha-hydroxy-1-dehydrotestosterone)(V). When II was used as a substrate, it was metabolized into III, IV, and V at a slower rate than I. Furthermore, 16alpha-hydroxy-androst-4-ene-3, 17-dione (16alpha-hydroxyandrostenedione)(X) was transformed into IV and V. Pregn-4-ene-3, 20-dione (progesterone)(XII) was transformed into androsta-1, 4-diene-3, 17-dione (androstadienedione)(VIII) and 17a-oxa-androsta-1, 4-diene-3, 17-dione (1-dehydrotestololactone)(IX), while 17alpha-hydroxy-pregn-4-ene-3, 20-dione (17alpha-hydroxyprogesterone)(VI) was converted into its 1-dehydro analogue (VII) without accumulation of any 20-dihydro compounds. Substrate specificity in the 20-reductase system of F. solani, Cylindrocarpon radicicola, Septomyxa affinis, Bacillus lentus, and three strains of B. sphaericus are demonstrated. The 20-reductase is active only on steroids having the 16alpha, 17alpha-oxido, and Delta(4)-3-keto functions. Evidence of competition between side-chain degrading enzymes and the 20-reductase for the steroid molecule and evidence of side-chain degradation followed by epoxide cleavage (and not the reverse) are presented. A mechanism for the epoxide opening by nongerminating spores of F. solani is postulated.

"Spore plate method" for transformation of steroids by fungal spores entrapped in silica gel g.

A new technique for investigating steroid biotransformations involving the use of glucose-treated Silica Gel G thin-layer chromatography plates spotted with fungal spores and steroid substrates is described. The conversion is followed by the detection and identification of steroid metabolites and is carried out on single plates by using the spores of different fungi. During the entire process, the spores remain on the original spots and microscopical examination revealed no germination. The method was successfully applied to as little as 30 mug of substrates, and a single plate could be used to detect the steroid metabolizing activity of spores of as many as 15 different cultures.