cytellin and 9-hydroxy-4-androstene-3-17-dione

The strain of Mycobacterium sp. VKM Ac-1817D forms 9alpha-hydroxy-androst-4-ene-3,17-dione (9-OH-AD) as a major product from sitosterol. The formation of 9-OH-AD was accompanied with its partial destruction due to residual steroid-1-dehydrogenase (St1DH) activity. The activity was found to be induced by androst-4-ene-3,17-dione (AD), while other intermediates of sitosterol oxidation did not influence 1(2)-dehydrogenation. The enzyme is located mainly in the cytosolic fraction. The cytosolic St1DH (dimer, M (r) approximately 58 kDa) was partially purified by ammonium sulfate fractionation, ion-exchange chromatography on DEAE-Sepharose and Phenyl-Sepharose, and gel filtration on Bio-Gel A-0.5M. It expressed the St1DH activity toward both AD and 9-OH-AD.

Topics: Ammonium Sulfate; Androstenedione; Chemical Fractionation; Chromatography, Gel; Chromatography, Ion Exchange; Cytosol; Enzyme Induction; Models, Biological; Molecular Structure; Molecular Weight; Mycobacterium; Oxidoreductases; Sitosterols

Mycobacterium sp. VKM Ac-1815D and its derivatives with altered resistance to antibacterial agents were able to produce androst-4-ene-3,17-dione (AD) as a major product from sitosterol. In this study, those strains were subjected to subsequent mutagenization by chemical agents and UV irradiation in combination with sitosterol selection pressure. The mutant Mycobacterium sp. 2-4 M was selected, being capable of producing 9alpha-hydroxyandrost-4-ene-3,17-dione (9-OH-AD) as a major product from sitosterol, with a 50% molar yield. Along with 9-OH-AD, both AD and 9alpha-hydroxylated metabolites with a partially degraded side-chain were formed from sitosterol by the mutant strain. The strain was unable to degrade 9-OH-AD, but degraded androsta-1,4-diene-3,17-dione (ADD), thus indicating a deficiency in steroid 1(2)-dehydrogenase and the presence of 9alpha-hydroxylase activity.

Topics: Androstadienes; Androstenedione; Biotransformation; Molecular Structure; Mutagenesis; Mycobacterium; Oxidoreductases; Selection, Genetic; Sitosterols

Using a special selection procedure, several mutants of Mycobacterium vaccae were isolated which were capable of converting sterols to 9 alpha-hydroxyandrost-4-ene-3,17-dion (9-OH-AD). Two mutants, Mycobacterium vaccae ZIMET 11052 and 11053, respectively, were further investigated. Strains of the species Mycobacterium fortuitum are mainly used for commercially obtaining 9-OH-AD from sterols. In contrast to the species Mycobacterium fortuitum the species Mycobacterium vaccae has not been reported to contain pathogenic strains. This seems an advantage for industrial application. Mutants with the ability of converting sterols to 9 alpha-hydroxysteroids have a defect in the steroid-1-dehydrogenase activity which is, however, only a partial one. The remaining activity may cause an undesirable degradation of the steroid nucleus. The steroid-1-dehydrogenase activity was tested using an assay developed by ATRAT (1986). We confirmed two apparently distinct steroid-1-dehydrogenases in Mycobacterium fortuitum NRRL B-8119 as reported by WOVCHA et al. (1979). One of them has an activity on androst-4-ene-3,17-dion (AD). The activity is increased by induction with sitosterol. The other one is active on 9-OH-AD. But Mycobacterium vaccae does not possess steroid-1-dehydrogenase activity on 9-OH-AD, and the AD specific steroid-1-dehydrogenase is not effected by sitosterol. The consequence is a high level of protection against steroid nucleus degradation yielding an effective accumulation of 9-OH-AD in fermentations with Mycobacterium vaccae mutants.

Topics: Androstenedione; Models, Biological; Mutagenesis; Mycobacterium; Oxidoreductases; Sitosterols; Sterols