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Evaluation of the fluorescent probes N ile R ed and 25‐NBD‐cholesterol as substrates for steroid‐converting oxidoreductases using pure enzymes and microorganisms
Author(s) -
Faletrov Yaroslav V.,
Frolova Nina S.,
Hlushko Hanna V.,
Rudaya Elena V.,
Edimecheva Irina P.,
Mauersberger Stephan,
Shkumatov Vladimir M.
Publication year - 2013
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.12265
Subject(s) - chemistry , cholesterol oxidase , nile red , steroid , stereochemistry , enzyme , biochemistry , fluorescence , physics , quantum mechanics , hormone
The fluorescent probes N ile R ed (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 ( CYP 17A1) (binding energy value of −8.3 kcal·mol −1 ). Recombinant S accharomyces cerevisiae and Y arrowia lipolytica , both expressing CYP 17A1, were found to catalyze the conversion of N ile R ed into two N ‐dealkylated derivatives. The conversion by the yeasts was shown to increase in the cases of coexpression of electron‐donating partners of CYP 17A1. The highest specific activity value (1.30 ± 0.02 min −1 ) was achieved for the strain Y. lipolytica DC 5, expressing CYP 17A1 and the yeast's NADPH ‐cytochrome P450 reductase. The dye was also metabolized by pure CYP 17A1 into the N‐dealkylated derivatives, and gave a type I difference spectrum when titrated into low‐spin CYP 17A1. Analogously, docking simulations demonstrated that 25‐ NBD ‐cholesterol binds into the active site of the microbial cholesterol oxidase ( CHOX ) from B revibacterium 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 μ m 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, P seudomonas aeruginosa , possessing CHOX s and CHDH s. 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 ( CYP 17A1) and bacteria ( CHOX and CHDH ), with fluorescence‐based techniques.