Evaluation of the fluorescent probes Nile Red and 25-NBD-cholesterol as substrates for steroid-converting oxidoreductases using pure enzymes and microorganisms

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 17α-hydroxylase/ 17,20-lyase (CYP17A1) (binding energy value of -8.3 kcal·mol ^-1). 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 ± 0.02 min^-1) 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 -5.6 kcal·mol^-1). 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 ± 5.9 μm and 0.66 ± 0.14 s^-1, 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. Docking simulations show that fluorescent substances Nile Red and 25-NBD-cholesterol fit well the substrate-binding sites of CYP17A1 and the cholesterol oxidase (CHOX), respectively. Recombinant yeasts, expressing CYP17A1, as well as pure CYP17A1 catalyze the Nile Red conversion into N-dealkylated derivatives. 25-NBD-cholesterol is converted into its 4-en-3-one derivative by the CHOX and cholesterol dehydrogenase as well as by bacteria Pseudomonas aeruginosa.