The effect of cell wall components on glycine-enhanced sterol side chain degradation to androstene derivatives by mycobacteria.

beta-Sitosterol side chain degradation by Mycobacterium sp. NRRL MB 3683 results in the formation of androstene derivatives and is increased in the presence of glycine. As the sterol transformation is carried out inside the cell, higher product accumulation could indicate faster diffusion of highly hydrophobic substrate through the cell wall permeability barrier. Cell wall preparations were obtained to analyse the effect of glycine on peptidoglycan components. Peptidoglycan is known to be the target for glycine action. In glycine-treated preparations, the molar ratio of diaminopimelic acid:muramic acid, the marker compounds of tetrapeptides and glycan strands respectively, was about 60% lower than in the control. This indicates a possible reduction in cross-linking between peptide units and the destruction of peptidoglycan. Unexpectedly, glycine also caused changes in the relative proportion of mycolic acids to other lipids occurring in the strain used for this study. The enhancement of beta-sitosterol side chain degradation is likely to result from disturbing the integrity of the cell wall components responsible for the permeability barrier in mycobacteria.

Effect of inhibitors of cell envelope synthesis on beta-sitosterol side chain degradation by Mycobacterium sp. NRRL MB 3683.

The role of the lipid bilayer and the peptidoglycan of the mycobacterial cell wall in the permeation of beta-sitosterol into the cell and its transformation to androst-1-ene-3,17-dione (AD) and androsta-1,4-diene-3,17-dione (ADD) was studied. Specific inhibitors were used at concentrations affecting the biosynthesis of the assumed target structures, but causing only partial cell growth inhibition or exerting no effect on growth. m-Fluorophenylalanine and DL-norleucine which are known to disorganize the biosynthesis of amphipatic components of the outer layer of the lipid bilayer, used at concentrations 250 micrograms/ml and 400 micrograms/ml, respectively, increased the formation rate of AD+ADD from 0.3 (control) to 0.7 and 0.8 mg products/g dry weight/h. The disorganization of the underlying mycolyl-arabinogalactan structure by the action of the ethambutol at the concentration 40 micrograms/ml, at which the cell growth was apparently not affected, caused the decrease of the product formation from 135 mg/l to 70 mg/l. In the presence of isoniazid (350 micrograms/ml) only trace amounts of AD accumulated during 48 hours of transformation indicating much lower activity than that of the intact cells. The most effective among the tested inhibitors of peptidoglycan synthesis were glycine (15 mg/ml) and vancomycin (150 micrograms/ml) which enhanced the transformation activity of the treated cells nearly three times. Increased transformation rate was also obtained by the action of colistin at concentrations ranging from 10 micrograms/ml to 15 micrograms/ml.

Elimination of by-products in 11 beta-hydroxylation of substance S using Curvularia lunata clones regenerated from NTG-treated protoplasts.

Stable mutants showing improved 11-hydroxylation of Substance S were isolated, following treatment with N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and regeneration of uninucleate protoplasts of the appropriate fungal strains. This procedure was especially suitable for obtaining more directed 11 beta-hydroxylation of Substance S with Curvularia lunata IM 2901. Apart from producing cortisol (11 beta-hydroxy-S), the parent strain formed several by-products that significantly lowered the yield of the desired 11 beta-hydroxyderivative. Isolated mutants of this microorganism carried out directed 11 beta-hydroxylation with only a small amount of one of the by-products, which resulted in a much higher yield of cortisol.

Stabilization of steroid 11-hydroxylation activity ofCunninghamella elegans protoplasts in organic osmotic stabilizers.

Protoplasts ofCunninghamella elegans, showing 11α-, and 11ß-hydroxylating ability of Substance S, preserved high transformation activity when dispersed in glucose-enriched, organic osmotic stabilizers. A joint action of polyoxins and 2-deoxy-D-glucose was necessary to prevent regeneration of the cell wall in long-lasting experiments. Stabilized and active, dispersed protoplasts may be an alternative research model for studying the function of the cell wall and intracellular metabolic pool constituents in steroid hydroxylation.

Determination of cytochrome P-450 in Cunninghamella elegans intact protoplasts and cell-free preparations capable of steroid hydroxylation.

Cytochrome P-450 was shown to be involved in 11 alpha-, and 11 beta-hydroxylation of Substance S in intact C. elegans protoplasts. The steroid transformation was inhibited by carbon monoxide, the inhibitory effect being dependent on CO concentration. The function of cyt P-450 in intact protoplasts was confirmed by the estimation of strong absorption at 450 nm in the CO difference spectrum. The presence of antimycin A was necessary to prevent the reduction of the cytochrome oxidase and its interference with the cyt P-450 in the spectrophotometric analysis. The intracellular content of cyt P-450 could be increased from 5.25 pM/mg protein to 26.88 pM/mg protein when the steroid inducer was present in the medium at each stage of protoplast preparation and during cyt P-450 determination. The enriched microsomal fraction obtained from the crude extract of ruptured protoplasts contained the steroid 11 alpha-hydroxylase system of C. elegans. The activity of 11 beta-hydroxylase could not be detected under the conditions of the experiment. The localization of steroid hydroxylases of C. elegans in microsomes was confirmed by cyt P-450 detection in the 9600 x g supernatant. Membranous fractions (pellets 1100 x g and 9600 x g) of the concanavaline A stabilized protoplasts, carrying the marker plasma-membrane-bound and mitochondrial ATPases, did not show maximum absorption at 450 nm in the CO difference spectrum.

Biotransformations of steroids.

Different types of microbiological transformation of steroids are reviewed, with special attention given to bioconversions applied in the manufacturing of steroid hormones, i.e., 11 alpha- 11 beta-, 16 alpha-, 17 alpha-hydroxylations and 1-dehydrogenation. Availability and utilization of raw materials for industrial production of steroids of the estrane, androstane, and pregnane series are discussed. Among the current trends in steroid research of a practical nature, immobilization of enzymes and living cells and the spore process are emphasized as alternative techniques of steroid transformation of possible future importance. Efforts to recognize, in cell-free preparations, the components of steroid-transforming enzyme systems as well as the cellular mechanisms of control of their biosynthesis and activity are described in order to illustrate the main subjects of current basic investigation in steroid bioconversion.

Protoplast release from fungi capable of steroid transformation.

Protoplasts were obtained from Hyphoderma roseum (Fries) and Cunninghamella elegans (Lendner), fungi capable of steroid 11-hydroxylation. The lytic enzyme preparation was derived from Trichoderma viride CBS 354-33. Homogeneous protoplast suspension, free of mycelial debris and cell wall fragments, transformed cortexolone and 6 alpha-fluorocortexolone-16,17-acetonide to the same products as the intact mycelium of the microorganisms. Liberation of protoplasts and their stabilization during steroid transformation was the most effective in 0.8 M MgSO4; still, this compound impaired steroid hydroxylation. Consequently, the concentration of the transformation product formed was nearly the same as in sucrose, mannitol, and sorbitol, compounds which caused no inhibition but which were less effective stabilizers.

Factors regulating the steroid 11-hydroxylation by non-germinating spores of Cunninghamella elegans (Lendner).

In the presence of malate or citrate sporangiospores of C. elegans were able to hydroxylate cortexolone with a rate twofold exceeding that of the control, water suspended spores. Analysis of the intracellular nicotinamide coenzyme pools revealed an increased NADPH:(NADP+ + NADPH) ratio, indicating more effective NADPH-generating systems in malate- or citrate-stimulating spores. Swollen spores remaining in the pregermination state, retained higher cortexolone-hydroxylating activity in the absence of malate and citrate. In these spores degradation of endogenous alanine and glutamic acid was observed. Possible NADPH-generating systems in C. elegans sporangiospores were discussed.

Regulation of steroid 16alpha-hydroxylation in Streptomyces olivoviridis.

The steroid 16alpha-hydroxylase of Streptomyces olivoviridis was shown to be constitutive, but synthetized only in small amounts in the absence of cortisol. The induction of the enzyme with the steroidal substrate resulted in a distinct increase in the 16alpha-hydroxylation rate. This increase in activity, however, could be demonstrated only in later phases of culturing, after the growth of the mycelium was completed. The possible mechanisms controlling the activity of the enzyme during the growth phase of the culture are discussed.

Adenylate energy charge and the steroid 11 alfa-hydroxylase activity in Monosporium olivaceum during growth and starvation.

During growth of Monosporium olivaceum its energy charge, E.C., (i.e. the adenylates ratio ATP + 0.5 ADP/ATP + ADP + AMP) increased from an initial value of 0.59 up to 0.85 after 25 hr of growth and then decreased to 0.51. The increase of energy charge was followed by the decrease of the activity of the 11 alpha-hydroxylase of cortexolone. This occured very clearly in the starved mycelium. Highest hydroxylation activity was observed when the lowest E.C. level (0.39-0.33) was reached.

Changes in the cellular content of the pool constituents of Monosporium olivaceum -- a steroid hydroxylating mould.

Changes of the metabolic pool constitutents of Monosporium olivaceum -- a mould capable of steroid hydroxylation were examined. The experiments were carried during growth and starvation of the microorganism. The highest activity of the 11alpha-hydroxylase was observed in the mycelium which contained the lowest level of free amino acids, glucose, and mannitol. It is suggested that the inhibition of biosynthetic processes and the decrease of the respiration rate, the activity of the NAD(P)H regenerating systems maintained, provide the optimal physiological conditions for the activity of the steroid hydroxylases.