Electrocatalytic hydroxylation of sterols by steroid C25 dehydrogenase from Sterolibacterium denitrificans

Abstract

We report electrochemically driven catalysis of the complex molybdoenzyme steroid C25 dehydrogenase (S25DH) from the β-Proteobacterium Sterolibacterium denitrificans. S25DH catalyzes 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-hydroxy-cholest-4-en-3-one as a product of catalytic turnover. Cholecalciferol (vitD3) is also a substrate. S25DH was immobilized on a modified Au working electrode with the co-adsorbent chitosan. The complexes ferricyanide ([Fe(CN)6]3-) 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 solubilizer in addition to 2-methoxyethanol. The catalytic activity varied depending upon the concentration of solubilizer in the reaction mixture. Parallel studies using ferricyanide as a chemical (as opposed to electrochemical) oxidant coupled to HPLC analysis show that S25DH is capable of oxidising both vitD3 and its less stable isomer pre-vitD3 and that the former substrate is stabilised by HPCD