title: Evaluation of the fluorescent probes Nile Red and 25-NBD-cholesterol as substrates for steroid-converting oxidoreductases using pure enzymes and microorganisms.
abstract: 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.
authors.affiliation: Research Institute for Physical Chemical Problems, Belarusian State University, Minsk, Belarus.
authors.forename: Yaroslav V
authors.initials: YV
authors.lastname: Faletrov
authors.forename: Nina S
authors.initials: NS
authors.lastname: Frolova
authors.forename: Hanna V
authors.initials: HV
authors.lastname: Hlushko
authors.forename: Elena V
authors.initials: EV
authors.lastname: Rudaya
authors.forename: Irina P
authors.initials: IP
authors.lastname: Edimecheva
authors.forename: Stephan
authors.initials: S
authors.lastname: Mauersberger
authors.forename: Vladimir M
authors.initials: VM
authors.lastname: Shkumatov
mesh_terms: D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D000478:Alkylation; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001951:Brevibacterium / Q000201:enzymology / Q000378:metabolism; D020134:Catalytic Domain; D002784:Cholesterol / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D002789:Cholesterol Oxidase / Q000737:chemistry / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D005656:Fungal Proteins / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008968:Molecular Conformation; D062105:Molecular Docking Simulation; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D010078:Oxazines / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013254:Steroid 17-alpha-Hydroxylase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: C115320:25-(N-((7-nitrobenz-2-oxa-1,3-diazol-4-yl)methyl)amino)-27-norcholesterol; D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D005656:Fungal Proteins; D010078:Oxazines; D011994:Recombinant Proteins; D002784:Cholesterol; D010088:Oxidoreductases; D002789:Cholesterol Oxidase; C501892:CYP17A1 protein, human; D013254:Steroid 17-alpha-Hydroxylase; C019884:cholesterol dehydrogenase; D009251:NADPH-Ferrihemoprotein Reductase; D009327:4-Chloro-7-nitrobenzofurazan; C044808:nile red