Katja Breskvar

A possible role of cytochrome P-450 in hydroxylation of progesterone by Rhizopus nigricans

Hydroxylation of progesterone was performed with intact cells, cell homogenate and with different subcellular fractions. The hydroxylating system in Rhizopus nigricans was found in post-mitochondrial supernatant. The reaction was inhibited by carbon monoxide and the inhibition was reversed by irradiation with light at a predominant wavelength of 450 nm thus indicating the involvement of cytochrome P-450 in the hydroxylation reaction. Using the reduced CO difference spectra, we have located the enzyme cytochrome P-450 in the membrane fraction sedimenting at 105.000 g.

Inducibility of cytochrome P-450 and of NADPH-cytochrome C reductase in progesterone treated filamenteous fungi Rhizopus nigricans and Rhizopus arrhizus☆

Abstract The inducibility of the enzymes involved in the 11α-hydroxylation of progesterone was studied in filamentous fungi Rhizopus nigricans and Rhizopus arrhizus . We showed that besides the cytochrome P-450, the NADPH cytochrome-C reductase is another component of this steroidal monooxygenase system. The inducibility of cytochrome P-450 and of the reductase was followed by measuring the level of both enzymes during the induction of the hydroxylase in the fungal mycelia with progesterone; neither cytochrome P-450 nor the reductase were present in measurable amounts in the noninduced fungi, whereas the level of both enzymes increased in parallel during the induction. The induction kinetics of the hydroxylase in the intact mycelia is very similar to the induction kinetics of the individual enzymes which was followed in the cell free preparations. It is concluded that the 11α-hydroxylation of progesterone in R. nigricans and R. arrhizus requires the induction, both, of cytochrome P-450 and of NADPH cytochrome-C reductase.

The role of cytochrome P45011α in detoxification of steroids in the filamentous fungus Rhizopus nigricans

Abstract The evidence was presented that steroid hydroxylating enzyme complex induced by substrate in the filamentous fungus Rhizopus nigricans (R. nigricans) alleviated toxic effect(s) of the steroid on fungal growth. The growth inhibition of fungal mycelium observed in steroid-containing culture(s) became much more obvious when fungal mycelia were grown in the simultaneous presence of inducing steroid and the P45011α inhibitor metyrapone. One the other hand, in experiments where we followed the fate of radioactively labelled progesterone added to the mycelial suspension, we noticed that steroid, after being initially accumulated in the microorganism, was, after some time, released from it; the latter phenomenon was not observed if induction of 11α-hydroxylase was prevented by cycloheximide. Results of experiments presented in this communication can be regarded as the first strong indication that the biological role of P45011α induction in R. nigricans is in removal of steroids which are toxic for the mycelium.

Inducibility of progesterone hydroxylating enzymes in Rhizopus nigricans

Abstract The induction of progesterone hydroxylases in Rhizopus nigricans was studied using chloramphenicol and cycloheximide as inhibitors of protein synthesis; effective inhibition of protein synthesis and enzyme induction was observed only with cycloheximide. Fungal mycelia not pretreated with the inducer do not hydroxylate progesterone if hydroxylation is assayed in the presence of cycloheximide indicating that the progesterone hydroxylating enzymes are inducible in this organism. Studies of the induction of progesterone hydroxylases in the growth medium and in the buffer solution revealed that the induction proceeds much faster in the buffer solution th

Resolution and reconstitution of cytochrome P-450 containing steroid hydroxylation system of Rhizopus nigricans

11 alpha-hydroxylation of progesterone in the eucaryotic filamentous fungus Rhizopus nigricans is catalyzed by a monooxygenase. Three components of this multienzyme system, cytochrome P-450, rhizoporedoxin and a FAD containing rhizoporedoxin reductase have been separated from the postmitochondrial fraction on DEAE cellulose. Using NADPH as electron donor we showed that the presence of all three components was necessary for the reconstitution of the active electron transport chain.

Localization of the gene encoding steroid hydroxylase cytochrome P-450 from Rhizopus nigricans inside a HindIII fragment of genomic DNA

The gene encoding steroid inducible cytochrome P450 of Rhizopus nigricans ATCC 6227b has been found inside a HindIII fragment of the genomic DNA by hybridization with a partial length cDNA probe. The latter was isolated by immunoscreening a cDNA library prepared in the lambda gt11 expression system and identified on the basis of inducibility and sequence analysis. The nucleotide sequence of the cDNA probe revealed a coding sequence for the heme binding segment characteristic of the P450 gene family.

Catalytic and immunochemical properties of NADPH-cytochrome P450 reductase from fungus Rhizopus nigricans.

Flavoprotein NADPH-cytochrome P450 reductase (CPR, EC 1.6.2.4) from filamentous fungus Rhizopus nigricans is a membrane bound enzyme which is involved in the reduction of cytochrome P450 during the hydroxylation of progesterone at 11alpha position. After purification of the enzyme from induced mycelia three forms of fungal CPR were detected on SDS-PAGE: a predominant form with an apparent molecular mass of 78kDa and two truncated forms. N-terminal sequences of all three forms were determined as well as some internal sequences of 78kDa form. Dose-dependent immunoinhibition of NADPH-cytochrome c reductase and progesterone 11alpha-hydroxylase activities was observed with mouse anti-CPR antisera. No cross-reactions were obtained on Western blots between mouse anti-CPR antisera and protein preparations from noninduced mycelia and microsomal fraction from fungus Pleurotus osteatus, plant Ginkgo biloba or chicken liver. The kinetic mechanism of CPR was proposed on the basis of model reaction with cytochrome c(3+). Results obtained at high ionic strength suggest a nonclassical two-site ping pong mechanism and at low ionic strength a sequential mechanism of bisubstrate reaction.

Resolution and reconstitution of the NADPH-cytochrome c (P-450) reductase induced by progesterone in Rhizopus nigricans.

The NADPH-cytochrome c (P-450) reductase induced in the filamentous fungus Rhizopus nigricans as a component of 11 alpha-hydroxylase of progesterone was resolved by DEAE-cellulose chromatography into two components. One of the components is an iron-sulfur protein (rhizoporedoxin), whereas the other component is a protein with reductase activity dependent on NADPH (rhizoporedoxin reductase). As shown in the reconstitution assay, the NADPH-cytochrome c (P-450) reductase activity was restored upon combination of these two proteins.

Solubilization and separation of microsomal cytochrome p-450 and NADPH-cytochrome C reductase of rhizopus nigricans

Abstract Microsomes from induced mycelia of R. nigRIcans solubilized with sodium deoxycholate contained cytochrome P-450 and NADPH-cytochrome c reductase. Complete separation of both enzymes was achieved by DEAE-cellulose ion exchange chromatography.

Specific interactions of steroids, arylhydrocarbons and flavonoids with progesterone receptors from the cytosol of the fungus Rhizopus nigricans

Rhizopus nigricans (R. nigricans) transforms fungitoxic progesterone into the less toxic 11alpha-hydroxyprogesterone which is then able to exit the mycelia into the surrounding water. Hydroxylation of progesterone is an inducible process in which cytosolic progesterone receptors could be involved. In the present study, we characterised receptors with respect to ligand specificity and to their involvement in progesterone induction of hydroxylase. EC(50) values of different ligands (steroids, xenobiotic arylhydrocarbons and natural flavonoids) were determined by competition studies using 40nM ((3)H)progesterone. C21 and C19 3-oxo-4-ene steroids were good competitors (EC(50) of progesterone 2.3 +/- 0.1 x 10(-7)M, EC(50) of androsten-3,17-dione 24 +/- 2 x 10(-7)M). The presence of hydroxyl groups in steroids significantly decreased the affinity for receptors. The arylhydrocarbons alpha-naphthoflavone and ketoconazole exhibited EC(50) values of 0.3 +/- 0.01 x 10(-7)M and 27 +/- 5 x 10(-7)M, respectively, whereas beta-naphthoflavone and benzo(a)pyrene were not able to displace labelled progesterone completely. The competition curves obtained by natural flavonoids also did not reach the bottom level of non-labelled progesterone, indicating the interaction at some allosteric binding site(s) of progesterone receptors. All ligands were examined for their involvement in progesterone-hydroxylase induction. Steroid agonists induced the enzyme in a dose-dependent manner in accordance with their affinity for receptors, whereas arylhydrocarbons and natural flavonoids did not induce the enzyme. The agonistic action of steroids, together with the antagonistic action of alpha-naphthoflavone, strongly suggests the involvement of progesterone receptors in progesterone signalling resulting in the induction of progesterone-hydroxylase.