The fluorescent probes Nile Red (nonsteroidal dye) and 25-{N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-methyl]amino}-27-norcholesterol (25-NBD-cholesterol) (a cholesterol analog) were evaluated as novel substrates for steroid-converting oxidoreductases. Docking simulations with AUTODOCK showed that Nile Red fits well into the substrate-binding site of cytochrome P450 17a-hydroxylase/17,20-lyase (CYP17A1) (binding energy value of À8.3 kcalÁmol À1 ). Recombinant Saccharomyces cerevisiae and Yarrowia lipolytica, both expressing CYP17A1, were found to catalyze the conversion of Nile Red into two N-dealkylated derivatives. The conversion by the yeasts was shown to increase in the cases of coexpression of electron-donating partners of CYP17A1. The highest specific activity value (1.30 AE 0.02 min À1 ) was achieved for the strain Y. lipolytica DC5, expressing CYP17A1 and the yeast's NADPH-cytochrome P450 reductase. The dye was also metabolized by pure CYP17A1 into the N-dealkylated derivatives, and gave a type I difference spectrum when titrated into low-spin CYP17A1. Analogously, docking simulations demonstrated that 25-NBD-cholesterol binds into the active site of the microbial cholesterol oxidase (CHOX) from Brevibacterium sterolicum (binding energy value of À5.6 kcalÁmol À1 ). The steroid was found to be converted into its 4-en-3-one derivative by CHOX (K m and k cat values were estimated to be 58.1 AE 5.9 lM and 0.66 AE 0.14 s À1 , respectively). The 4-en-3-one derivative was also detected as the product of 25-NBD-cholesterol oxidation with both pure microbial cholesterol dehydrogenase (CHDH) and a pathogenic bacterium, Pseudomonas aeruginosa, possessing CHOXs and CHDHs. These results provide novel opportunities for investigation of the structure-function relationships of the aforementioned oxidoreductases, which catalyze essential steps of steroid bioconversion in mammals (CYP17A1) and bacteria (CHOX and CHDH), with fluorescence-based techniques. -methyl]amino}-27-norcholesterol; ABC, ATP-binding cassette; AdR, adrenodoxin reductase; Adx, adrenodoxin; CHDH, microbial cholesterol dehydrogenase; CHOX, microbial cholesterol oxidase; Nile Red, 9-diethylamino-5H-benzo[alpha]phenoxazine-5-one; CPR, NADPH-cytochrome P450 reductase; CYP, cytochrome P450; CYP17A1, cytochrome P450 17a-hydroxylase/17,20-lyase; HPLC-FL, HPLC with fluorometric detection; HSD, hydroxysteroid dehydrogenase; PDB, Protein Data Bank.
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