title: 22-NBD-cholesterol as a novel fluorescent substrate for cholesterol-converting oxidoreductases.
abstract: Docking simulations and experimental data indicate that 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3β-ol (22-NBD-cholesterol), a common fluorescent sterol analog, binds into active sites of bovine cytochrome P450scc and microbial cholesterol dehydrogenases (CHDHs) and then undergoes regiospecific oxidations by these enzymes. The P450scc-dependent system was established to realize N-dealkylation activity toward 22-NBD-cholesterol, resulting in 7-nitrobenz[c][1,2,5]oxadiazole-4-amine (NBD-NH(2)) formation as a dominant fluorescent product. Basing on LC-MS data of the probes derivatized with hydroxylamine or cholesterol oxidase, both pregnenolone and 20-formyl-pregn-5-en-3β-ol were deduced to be steroidal co-products of NBD-NH(2), indicating intricate character of the reaction. Products of CHDH-mediated conversions of 22-NBD-cholesterol were defined as 3-oxo-4-en and 3-oxo-5-en derivatives of the steroid. Moreover, the 3-oxo-4-en derivative was also found to be formed after 22-NBD-cholesterol incubation with pathogenic bacterium Pseudomonas aeruginosa, indicating a possible application of the reaction for a selective and sensitive detection of some microbes. The 3-keto-4-en derivative of 22-NBD-cholesterol may be also suitable as a new fluorescent probe for steroid hormone-binding enzymes or receptors.
authors.affiliation: Research Institute for Physical Chemical Problems, Belarusian State University, Leningradskaya str 14, 220030 Minsk, Belarus. yaroslav82@tut.by
authors.forename: Yaroslav V
authors.initials: YV
authors.lastname: Faletrov
authors.forename: Katsiarina I
authors.initials: KI
authors.lastname: Bialevich
authors.forename: Irina P
authors.initials: IP
authors.lastname: Edimecheva
authors.forename: Dzmitry G
authors.initials: DG
authors.lastname: Kostsin
authors.forename: Elena V
authors.initials: EV
authors.lastname: Rudaya
authors.forename: Ekaterina I
authors.initials: EI
authors.lastname: Slobozhanina
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; D000818:Animals; D002417:Cattle; D002784:Cholesterol / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D002789:Cholesterol Oxidase / Q000378:metabolism; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D062105:Molecular Docking Simulation; D009615:Nocardia / Q000201:enzymology* / Q000378:metabolism; D010088:Oxidoreductases / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: C077527:7-nitrobenz-2-oxa-1,3-diazol-4-ylcholesterol; D005456:Fluorescent Dyes; D002784:Cholesterol; D010088:Oxidoreductases; D002789:Cholesterol Oxidase; D002786:Cholesterol Side-Chain Cleavage Enzyme; C019884:cholesterol dehydrogenase; D009327:4-Chloro-7-nitrobenzofurazan