Steroidumwandelnde Enzyme aus Mikroorganismen XII. Merkmale der Induktion der 4‐En‐3‐oxosteroid: (Akzeptor)‐l‐en‐oxidoreduktase in <i>Nocardia opaca</i>

The steroid 16a-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 t h e 16a-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 t h e growth phase of t h e culture are discussed.The ability of certain streptomycetes to 16 a-hydroxylate the steroid nucleus was first described by PERLMAN et al. in In previous work less attention was paid to the mechanisms of regulation of steroidhydroxylase activity. In our present paper more detailed studies on the relationship between cell growth, intracellular metabolic pool constituents, and the 16 a-hydroxylation activity were carried out. Materials and methodsMicroorganism: A streptomycete strain, isolated from t h e local soil on grounds of its ability for 16a-hydroxylation of C-21 steroids was used. The choice was dictated by t h e practically onedirectional transformation of hydrocortisone. The strain was identified as Streptomyces olivoviridis at t h e Department of Bacteriology of the State Institute of Hygiene, Warsaw.Media: The stock culture was kept on slants of Bacto-Potato-Dextrose-Agar (DIFCO) a t 4 "C and reinoculated once a month. For conidia germination a medium (designated as medium 1 a ) of the following composition was used (g/l): corn steep liquor 50% -20, sucrose -30, (NH,),SO, --2, CaCO, -7.Growth and bioconversion were investigated on medium D of following composition (g/l) : soluble starch -40, corn steep liquor 5076 -20, CaCO, -5 (MARTINKOVA and DYR 1965). The media were sterilized at 117 "C for 30 min. After sterilization the media were adjusted to p H = 6.7.Reagents : Hydrocortisone (SCHERING) was the steroid substrate. The product of hydroxylation was compared with 1%-hydroxyhydro-cortisone (POLFA). The substrate was dissolved in N,Ndimethylformamide (KOCH-LIGHT Lab.). Sugar and amino acids were purchased from MERCK and CALBIOCHEM, respectively.Cultivation and bioconversion: Conidia were washed off from a 14-day culture on potato slants and t h e suspension was transferred to 20 ml of medium l a in 200-ml-Erlenmeyer flasks. Incu-

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Regulation of steroid 16α‐hydroxylation in Streptomyces olivoviridis

Długoński

Sedlaczek

1981

Steroidumwandelnde Enzyme aus Mikroorganismen. XIII. Beziehungen zwischen Steroidstruktur und Induktion der 4‐En‐3‐oxosteroid: (Akzeptor)‐l‐en‐oxidoreduktase in Nocardia opaca

Hörhold

Hüller

Rose

1980

The induction of the 4-ene-3-oxosteroid: (acceptor)-1-ene-oxidoreductase in Nocardia opaca has been shown to be dependent on both hydrophilic and hydrophobic structural features of the steroid molecule, i.g. the nature of the oxygen function in positions 11 and 20 and the presence of the methyl group in position 10 influence the induction of the enzyme drastically. In this connection the metabolism of the steroid inductors being degradated during the induction process has been considered.

Factors regulating the steroid 11‐hydroxylation by non‐germinating spores of Cunninghamella elegans (LENDNER)

Jaworski

Sedlaczek

Wilmańska

et al. 1982

I n 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 a n increased NADPH : (NADP+ + NADPH) ratio, indicating more effective NADPH-generating systems in malateor citrate-stimulating spores.Swollen spores remaining in the pregermination state, retained higher cortexolone-hydroxylating activity in the absence of malate and citrate. I n these spores degradation of endogenous alanine and glutamic acid was observed. Possible NADPH-generating systems in C. elegans sporangiospores were discussed. Among many factors controlling microbiological transformation of steroids ( H~R - HOLD et al. 1979, 1980, DLUGONSKI and SEDLACZEK 1981 an important role seems to be played by systems producing reduced nicotinamide-adenine dinucleotides. These coenzymes are present in microorganisms in low concentration, and, since they participate in basic processes of primary metabolism like respiration and biosynthesis, other NAD( P)H-dependent enzyme systems are effectively controlled in vivo by the accessibility of these cofactors.There a number of enzymes is known whose activity is dependent on the level of nicotinamide nucleotides concentration or on the ratio of their oxidized and reduced forms (BARNES et al. 1972, DUNN-COLEMAN and PATEMAN 1977).This kind of relationship was also reported for lla-steroid hydroxylase ( SEDLACZEK et nl. 1979). A particularly useful model to determine the correlation between the activity of steroid-transforming enzyme and the level of a given metabolite is provided by eonie fungal spores. Retaining their steroid conversion ability, the spores show much lower metabolic activity than the mycelium. Owing to this, the process in question is less affected by side reactions.Our earlier report (SEDLACZEK et al. 1981) revealed that the rate of cortexolone hydroxylation by mycelium of C. elegans was twice greater than that of sporangiospores. The increased activity was acconipanied by the degradation of the intracellular free amino acids, notably of alanine and glutaniic acid and by the higher level of the NAI)P+ + NBDPH pool reduction.The aim of our present work was to stimulate the hydroxylation of cortexolone by C. elegans sporangiospores and to investigate the changes of the nicotinamide nucleotides and amino acids in the stimulated spores. Materials and methodsMicroorganism: A strain of C. elegans (LENDNER) isolated in our laboratory from a n African soil sample, identified in the Department of Botany, University of Warsaw, was used.