19 Acta Chim. Slov. 1998, Y5(1), pp. 19-25 (Received 28.1.1998) SUBSTRATE SPECIFICITY OF 17ß-HYDROXYSTEROID DEHYDROGENASE FROM PLEUROTUS OSTREATUS M. Pogačar, M. Zorko and M. Zakelj-Mavric Institute of Biochemistry, Medical Faculty, Ljubljana, Slovenia ABSTRACT We present evidence which suggests that Pleurotus ostreatus 17ß-HSD is a pluripotent enzyme which can oxidize 17ß-hydroxysteroids and even more so hydroquinone in the presence of NAD+. The study of the reverse reaction indicates that the carbonyl reductase activity prevails over the hydroxysteroid dehydrogenase activity. Kinetic studies also reveal the presence of a separate acetoacetyl CoA reductase / ß-hydroxybutyryl CoA dehydrogenase activity in Pleurotus ostreatus. INTRODUCTION The metabolism of xenobiotics and steroids has been intensively studied from the point of view of their oxidation by different cytochromes P-450. Recently, the reduction of these compounds by carbonyl reducing enzymes has attracted more attention. A group of enzymes, classified either as hydroxysteroid dehydrogenases (HSDs) or carbonyl reductases, was found to exhibit specificity towards both groups of compounds (1). 17ß-hydroxysteroid dehydrogenases (17ß-HSDs) can be taken as representatives of this group of enzymes since several 17ß-HSDs from mammalian peripheral as well as steroidogenic tissues have been found to posses carbonyl reductase activity in addition to 20 hydroxysteroid dehydrogenase activity (2-7). In fungi the pluripotency has been established only for 17b-HSD from the filamentous Cochliobolus lunatus (8). The white-rot fungus Pleurotus ostreatus has so far been found to metabolize polycyclic aromatic hydrocarbons (9) as well as to oxidize androgens and estrogens (10). Since recently a preliminary study suggested several differences in the characteristics between 17b-HSDs from both fungi, we studied Pleurotus ostreatus 17b-HSD in greater detail. EXPERIMENTAL 1) Fungal species Pleurotus ostreatus G7 was obtained from the Microbial Culture Collection of the Chemical Institute , MZKIBK Ljubljana. 2) Growth conditions Ten-day-old cultures on agar slants ( 2% agar in 60 Blg malt extract) were used to inoculate 100 ml of liquid media composed of 0.5% cornstep liquor, 1% oatmeal, 1% glucose, 4% tomato paste, 10ml/l mineral solution, pH 7 ( mineral solution: 1g/l FeSO4 x 7H2O, 1g/l MnSO4 x0 H2O, 0.2 g/l ZnSO4 x 7H2O, 0.1 g/l CaCl2 x 2H2O, 0.056 g/l H3BO3, 0.0025 g/l CuC03, 0.019 g/l (NH4)6Mo7O24 x 4H2O). Cultivation was performed in 500 ml Erlenmeyer flasks for six days at 250 C on a rotary shaker at 110 rpm. 3) Enzyme preparation After six days of growth the mycelium of Pleurotus ostreatus was filtered and frozen by liquid nitrogen. The enzyme preparation used for kinetic measurements was prepared in 50 mM Tris/HCl, pH 9, 20% glycerol, as described previously (11). 4) Enzyme assays a) Chromatographic method During the partial purification procedure the enzyme activity was tested as described (11). b) Spectrophotometric method For the kinetic measurements a similar procedure was used as already described (8). 21 Measurements were performed against blanks without the substrate in the reaction mixture, or without the enzyme in the case of quinone. Reaction rates were expressed as relative enzyme activities (%) with different substrates oxidized or reduced by 100ml of the enzyme preparation. The enzyme activities with 100 mM testosterone and 100 mM NAD+ in 50 mM Tris/HCl, pH 8.5 with 20 % glycerol for the oxidation or 100 mM androstenedione and 100 mM NADH in 50 mM Tris/HCl, pH 7.0 with 20 % glycerol for the reduction reaction were taken as 100 %. 5) Competition between different substrates Competition between the most effective substrates was studied for the oxidation reaction because of more comparable relative activities of selected substrates and because of higher initial velocities of testosterone oxidation in comparison to androstenedione reduction. It was tested by following the activity of 17b-HSD in the presence of each individual substrate and in the presence of two substrates simultaneously; the concentration of one substrate varied (testosterone from zero to 250 mM, b-hydroxybutyryl CoA from zero to 200mM, and hydroquinone from zero to 400 mM ) while the concentration of the other was kept constant (testosterone 100mM, hydroquinone 150 mM and b-hydroxybutyryl CoA 75mM), and vice versa. The concentration of the enzyme was constant in all experiments. From the Michaelis-Menten curves fitted to the experimental results obtained in the presence of each individual substrate, we determined the apparent Km and Vmax for each individual substrate in the presence of 20 mM NAD+. These parameters were then used for the calculation of the curves valid for the competition between two substrates for the same enzyme or/and for the parallel action of two different enzymes on two substrates (12). RESULTS A range of hydroxylated steroid and non-steroid compounds was used to investigate the substrate specificity of the 17b-HSD enzyme preparation. Table 1 shows that it has a broad specificity for hydroxylated substrates with NAD+ required as a coenzyme. 22 Table 1. Relative oxidative activities of 17b-HSD enzyme preparation in the presence of different substrates and 100 mM coenzymes. The activities are normalized to the activity in the presence of 100 mM testosterone and 100 mM NAD+, considered to be 100%. SUBSTRATE CONC. COENZYME RELATIVE ACTIVITIES (%) testosterone 100jiM NAD+ NADP+ 100 0 estradiol 100jiM NAD+ NADP+ 80 0 hydroquinone 100jiM NAD+ NADP+ 116 0 DL- ß -hydroxybutyryl CoA 100jiM NAD+ NADP+ 506 0 L-ß-hydroxybutyrate 1mM NAD+ NADP+ 13 0 p-nitrobenzyl alcohol 1mM NAD+ NADP+ 69 0 L-malate 1mM NAD+ NADP+ 40 0 0 = no detectable activity b-Hydroxybutyryl CoA was found to be the best substrate while hydroquinone, testosterone, and estradiol were also readily oxidized. The relative activities for b-hydroxybutyrate, malate and p-nitrobenzyl alcohol were much smaller than for testosterone (100%). No enzyme activity could be detected in the presence of NADP+. In addition carbonyl compounds, including steroids, quinones, aromatic aldehyde and aliphatic ketones were tested as substrates for the reductase activity of 17b-HSD enzyme preparation (Table 2). Both nicotinamide nucleotides could serve as electron donors. Acetoacetyl CoA and benzoquinone were reduced at much higher relative activities than androstenedione in the presence of NADH (100%). No activity could be detected in the presence of the other tested compounds. 23 Table 2. Relative reductive activities of 17b-HSD enzyme preparation in the presence of different substrates and 100 mM coenzymes. The activities are normalized to the activity of 100 mM androstenedione and 100 mM NADH which was taken as 100%. SUBSTRATE CONC. COENZYME RELATIVE ACTIVITIES (%) androstenedione 100jiM NADH NADPH 100 31.3 estrone 100jiM NADH NADPH 0 0 benzoquinone 100jiM NADH NADPH 3103 3737 acetoacetyl CoA 100jiM NADH NADPH 3108 728 acetoacetate 1mM NADH NADPH 0 0 p-nitrobenzaldehyde 1mM NADH NADPH 0 0 0 = no detectable activity As the above mentioned results suggested that at least some of the reactions could be catalyzed by 17b-HSD, competition experiments between the most effective substrates testosterone, hydroquinone, and b-hydroxybutyryl CoA were performed. Testosterone was found to compete with hydroquinone for the active centre of 17b-HSD (Fig.1A). On the other hand, the results on Fig.1B suggest parallel oxidations of testosterone and b-hydroxybutyryl CoA catalyzed by two different enzymes. These results were confirmed by experimental data suggesting parallel oxidations also for hydroquinone and b-hydroxybutyryl CoA (data not shown). 24 A 400 30 200 100 300 [testosterone] , mM 400 300 200 100 300 [testosterone] , mM Fig.1. A: Competition between substrates testosterone (variable concentration) and hydroquinone (150 mM) for the active centre of 17b-HSD. B: Parallel oxidation of testosterone (variable concentration) and b-hydroxybutyrylCoA (75 mM) catalyzed by two different enzymes. Points were obtained experimentally but the curves were calculated as described in Experimental, section 5. CONCLUSIONS While the important role of 17b-HSD in mammalian organisms is well established (13) the question about the role of these enzymes in primitive eukaryotes is not yet clear. It represents a challenge to those interested in fungal metabolism per se as well as the evolution of HSD and steroid hormone signalling system. In this sense, further characterization of fungal 17b-HSD seems desirable. In the present study, the Pleurotus osteratus 17b-HSD enzyme preparation was found to have a broad substrate specificity catalyzing efficiently the oxidation of the steroid hormones testosterone and estradiol as well as the non-steroidal compounds hydroquinone and b-hydroxybutyryl CoA . NAD+ is required as an electron donor. The results of the reverse reaction revealed the prevailing carbonyl reductase and acetoacetyl 25 reductase activity over 17ß-HSD (reductase) activity. The competition experiments between the most effective substrates of 17ß-HSD enzyme preparation suggested the presence of two separate enzymes, 17ß-HSD and ß-hydroxybutyryl Co A dehydrogenase / acetoacetyl Co A reductase. Pleurotus osteratus 17ß-HSD was found to be pluripotent enzyme capable of testosterone and hydroquinone oxidation. It thus joins pluripotent HSDs whose role in detoxification of xenobiotic carbonyl compounds in addition to their role in the metabolism of endogenous steroids and quinones is only suspected (14). ACKNOWLEDGMENTS The authors wish to thank M.Marušič for skilful technical assistance. The work was supported by the Ministry of Science and Technology of Slovenia. REFERENCES (1) E. Maser, Biochem.Pharmacol. 1995, 49, 421-440. (2) R. Jarabak et al., Arch.Biochem.Biophys. 1996, 327, 174-180. (3) Y. Deyashiki et al., J.Biol.Chem. 1995, 270, 10461-10467. (4) T.Nishinaka et al., Enzyme 1992,46,221-228. (5) J.Klein et al., Eur. J.Biochem. 1992, 205, 1155-1162. (6) A.Hara et al., Arch.Biochem.Biophys. 1986, 249, 225-236. (7) H.Sawada et al., Biochem.Pharmacol. 1988, 37, 453-458. (8) T. Lanišnik Rižner et al., in Enzymology and Molecular Biology of Carbonyl Metabolism 6, ed.by Weiner et al., Plenum Press, New York, 1996, pp.569-577. (9) L. Bezalel et al., Appl.Environ.Microbiol. 1996, 62, 292-295. (10) T. Lanišnik et al., FEMSMicrobiol.Lett. 1992, 99, 49 -52. (11) T. Lanišnik Rižner et al., J.SteroidBiochem.Molec.Biol. 1996, 59, 205-214. (12) A. Cornish-Bowden, Fundamentals of Enzyme Kinetics, Portland Press, London, 1995, pp 108-109. (13) T.M. Penning, Endocrine Reviews 1997, 18, 281-305. (14) N. Iwata et al., J.Biochem. 1989, 105, 556-564. POVZETEK Na osnovi kinetičnih študij, prikazanih v članku, lahko sklepamo, da je 17ß-HSD iz glive Pleurotus ostreatus pluripotenten encim, ki lahko oksidira 17ß-hidroksisteroide in hidrokinon v prisotnosti NAD+. V smeri redukcije prevladuje karbonil-reduktazna aktivnost nad hidroksisteroidno reduktazno aktivnostjo. Rezultati kažejo, da je v glivi Pleurotus ostreatus prisotna tudi neodvisna acetoacetil CoA reduktaza / ß-hidroksibutiril CoA dehidrogenaza.