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 (Km and kcat 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.