22-NBD-cholesterol, a new fluorescent substrate of bacterial cholesterol-oxidases

Abstract

Fluorescent analogs of sterols are convenient agents for studying the transport, distribution, and protein-ligand interaction of natural sterols in cells [1]. However, the effect of the fluorescent moiety on the interaction parameters with cholesterol-transforming enzymes such as bacterial cholesterol-oxidases or mammalian steroid-hydroxylating cytochrome P450 has not been addressed in the literature. Herein we report data for the interaction of the fluorescent cholesterol analog 22-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-23,24-bisnor-5-cholen-3E-ol [22-NBD-cholesterol (1)] with cholesteroloxidases (EC 1.1.3.6). Cholesterol-oxidases are FAD-dependent enzymes that catalyze the transformation of 3E-hydroxy-' 5 -steroids and are widely used for laboratory and clinical cholesterol determinations [2].We calculated the interaction of 1 with the enzyme based on the three-dimensional structure of Brevibacterium sterolicum cholesterol-oxidase (code PDB 1COY) using the Autodock 4.0 programs and MGLTools. It was found with the help of molecular docking that 1 can bind to the active center of cholesterol-oxidase such that its 3E-hydroxy group is situated in close proximity to the functionally important His447 and Glu361 residues [3] and the redox-active groups of the FAD molecule. The calculated binding energies for cholesterol and 1 were practically the same (-11.43 and -11.94 kcal/mol, respectively).Next selective transformation of 1 was demonstrated using cholesterol-oxidase in vitro. After biotransformation of fluorescent substrate 1 with purified cholesterol-oxidase, the formation of 2, which retained the fluorescent properties of 1 (Scheme 1) was detected by HPLC (Shimadzu LC10-AD chromatograph with an RF-10Au1 fluorimetric detector and SPD-10A spectrophotometric detector; CH 3 CN:H 2 O:i-PrOH, 84:16:5; LiChroCART C18 column, Merck, 250 u 4 mm).A characteristic difference of the absorption spectra of product 2 from that of the substrate was the presence in it of a distinct O max (EtOH) at 240 nm. The mass spectrum (Shimadzu QP2010+ spectrometer, EI, 70 eV) of starting 1 contained [M] + at m/z 494 whereas the mass spectrum of 2 had [M] + with m/z 492. Formation of 2 was confirmed definitively by a characteristic singlet for vinyl proton H-4 (5.74, br.s) in the PMR spectrum (Bruker, 400 MHz, CDCl 3 ) that is present for other steroids with a conjugated ' 4 -3-keto structure [4,5]. Thus, the 3E-hydroxy-' 5 -group of 1 was transformed into a conjugated

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

“…Kinetic parameters of the CHOX-driven 25-NBDcholesterol oxidation, together with similar data for 22-NBD-cholesterol and cholesterol, are summarized in Table 2. As shown, k cat /K m values decreased in accordance with increasing volume and planarity of the steroids' side chains (substituents at C17) [22]. This is in agreement with the docking results.…”

“…6C). These data, together with our previous results concerning 22-NBD-cholesterol oxidation by CHOXs [22], confirm that the product is the 4-en-3-one derivative of 25-NBD-cholesterol (formally, '25-NBD-cholest-4-en-3-one') ( Fig. 6D).…”

“…). The calculated binding energy value for 25‐NBD‐cholesterol interaction was shown to be approximately −5.6 kcal·mol −1 , whereas for 22‐NBD‐cholesterol it was −11.9 kcal·mol −1 , indicating worse affinity for the CHOX of the former steroid than of the latter one with a shorter side chain. In the complex, the 7‐nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl group of 25‐NBD‐cholesterol is primarily surrounded by hydrophobic residues, comprising Ile85, Gly84, Phe83, Met59, and Leu60, which is a prerequisite for an increase in steroid fluorescence during its binding to the CHOX.…”

“…5). The calculated binding energy value for 25-NBD-cholesterol interaction was shown to be approximately À5.6 kcalÁmol À1 , whereas for 22-NBD-cholesterol it was À11.9 kcalÁmol À1 [22], indicating worse affinity for the CHOX of the former steroid than of the latter one with a shorter side chain.…”

“…Thus, the possibilities of enzymatic conversions of both 25-NBDcholesterol and Nile Red seem not to have been fully investigated. In general, only a few fluorescent substrates for steroid-converting oxidoreductases have been discovered [18][19][20][21][22][23]. Hence, the aim of this study was to evaluate Nile Red and 25-NBD-cholesterol as new fluorescent substrates for the steroid-converting oxidoreductases mentioned above, i.e.…”