Electrocatalytic Hydroxylation of Sterols by Steroid C25 Dehydrogenase from Sterolibacterium denitrificans

Abstract

The electrochemically driven catalysis of the complex molybdoenzyme steroid C25 dehydrogenase (S25DH) from the β-Proteobacterium Sterolibacterium denitrificans is reported. S25DH catalyses the oxygen-independent regioselective hydroxylation of the tertiary C25 atom of sterols and also their derivatives. Cholest-4-en-3-one is a native substrate for S25DH, which produces 25-hydroxycholest-4-en-3-one as a product of catalytic turnover. Cholecalciferol (vitD ) is also a substrate. S25DH was immobilised on a modified gold working electrode with the co-adsorbent chitosan. The complexes ferricyanide ([Fe(CN) ] ) and ferrocenium methanol (FM ) are effective artificial electron acceptors from S25DH and act as mediators of electron transfer between the electrode and the enzyme. 2-Hydroxypropyl-β-cyclodextrin (HPCD) was employed as a sterol solubiliser, in addition to 2-methoxyethanol. The catalytic activity varied, depending upon the concentration of solubiliser in the reaction mixture. Parallel studies with [Fe(CN) ] as a chemical (as opposed to electrochemical) oxidant coupled to HPLC analysis show that S25DH is capable of oxidising both vitD and its less stable isomer, pre-vitD , and that the former substrate is stabilised by HPCD.

Bacterial steroid hydroxylases: enzyme classes, their functions and comparison of their catalytic mechanisms

“…Warnke et al reported a more than threefold higher V max for VD3 (1.48 nmol/min/mg) compared to that obtained for the 3-hydroxy steroid 7-dehydrocholesterol (0.47 nmol/min/mg) (Warnke et al 2016 ). Recently, the apparent K M values were determined in an electrochemical system yielding K M = 740 μM for cholest-4-en-3-one and K M = 327 μM for VD3 (Kalimuthu et al 2018 ). The advent of overexpression system for S25DH in T. aromatic K172 allowed kinetic characterization of individual enzymes.…”

“…The proposed system was also applied for hydroxylation of various 3-ketosteroids such as: cholest-4-en-3-one, cholest-1,4-dien-3-one and cholest-4,6-dien-3-one with a final concentration in the range of 2.2 g/L, or sterols such as: cholesterol, 7-dehydrocholesterol, with a final concentration in the range of 0.8 g/L (Rugor et al 2017a ). Purified S25DH was also efficiently immobilized on silica (Rugor et al 2017a ) or on a microfiltration membrane (Kalimuthu et al 2018 ). In both cases, the enzymatic reaction was coupled to an electrochemical system, which turned out to be sensitive to changes in substrate concentration.…”

“…In both cases, the enzymatic reaction was coupled to an electrochemical system, which turned out to be sensitive to changes in substrate concentration. Due to that fact S25DH can be considered as a potential element for construction of an electrochemical biosensor detecting cholecalciferol or 3-ketosteroids as an electrochemical signal (current produced in the electrode reaction coupled to the enzymatic process) differs for different concentrations of steroids in the medium (Kalimuthu et al 2018 ).…”