Characterization of fungal 17beta-hydroxysteroid dehydrogenases.
To promote understanding of the evolution of the steroid hormone signalling and hydroxysteroid dehydrogenases (HSDs), comparative characterization of fungal 17beta-HSDs was performed. Constitutive 17beta-HSD activity was determined in cytosols of the fungi: Cochliobolus lunatus, Pleospora herbarum, Fusarium lini, Trichoderma viride, Mucor spinosus, Rhizopus nigricans and Pleurotus ostreatus. The reaction equilibrium in all species except P. ostreatus was shifted towards reduction. The preferential coenzyme for reduction of androstenedione was NADPH, while for oxidation of testosterone, NAD4 was preferred. The highest enzyme activities were found in the Ascomycete C. lunatus (152.4 nmol mg(-1) h(-1)) and in the Basidiomycete P. ostreatus (69.1 nmol mg(-1) h(-1)). No similarities on the protein and mRNA level between fungal 17beta-HSDs and the purified enzyme from C. lunatus were observed. To investigate the nature of these enzymes, 17beta-HSD was purified from P. ostreatus using ammonium sulphate precipitation, hydrophobic interaction chromatography, and affinity chromatography. The purified enzyme has an apparent molecular mass of approximately 35 kDa and is probably a dimer as determined by gel filtration. Chemical modifications exposed Lys, His and Tyr as important for enzyme activity. Additionally, no similarities of C. lunatus and P. ostreatus enzymes were found to bacterial 3alpha,20beta-HSD from Streptomyces hydrogenans, 3beta,17beta-HSD from Comamonas testosteroni and mammalian 17beta-HSD types 1 and 4. The results thus suggest that there are most probably different enzymes responsible for 17beta-HSD activity in filamentous fungi.
Rizner
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Institute of Biochemistry, Medical Faculty, University of Ljubljana, Slovenia. lanisnik@ibmi.mf.uni-lj.si
Zakelj-Mavric
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D015067:17-Hydroxysteroid Dehydrogenases / Q000737:chemistry* / Q000302:isolation & purification*; D000645:Ammonium Sulfate / Q000494:pharmacology; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002845:Chromatography; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D005658:Fungi / Q000201:enzymology*; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism
D012333:RNA, Messenger; D009243:NAD; D009249:NADP; D015067:17-Hydroxysteroid Dehydrogenases; C044256:3 (or 17)-beta-hydroxysteroid dehydrogenase; D000645:Ammonium Sulfate