Bacterial cholesterol oxidases are able to act as flavoprotein‐linked ketosteroid monooxygenases that catalyse the hydroxylation of cholesterol to 4‐cholesten‐6‐ol‐3‐one

Abstract

A new metabolite of cholesterol was found in reaction mixtures containing cholesterol or 4-cholesten-3-one as a substrate and extra- or intracellular protein extracts from recombinant Streptomyces lividans and Escherichia coli strains carrying cloned DNA fragments of Streptomyces sp. SA-COO, the producer of Streptomyces cholesterol oxidase. The new metabolite was identified as 4-cholesten-6-ol-3-one based on comparisons of its high-performance liquid chromatography, gas chromatography/mass spectrometry, infrared and proton-nuclear magnetic resonance spectra with those of an authentic standard. Genetic analyses showed that the enzyme responsible for the production of 4-cholesten-6-ol-3-one is cholesterol oxidase encoded by the choA gene. Commercially purified cholesterol oxidase (EC 1.1.3.6.) of a Streptomyces sp., as well as of Brevibacterium sterolicum and a Pseudomonas sp., and a highly purified recombinant Streptomyces cholesterol oxidase were also able to catalyse the 6-hydroxylation reaction. Hydrogen peroxide accumulating in the reaction mixtures as a consequence of the 3 beta-hydroxysteroid oxidase activity of the enzyme was shown to have no role in the formation of the 6-hydroxylated derivative. We propose a possible scheme of a branched reaction pathway for the concurrent formation of 4-cholesten-3-one and 4-cholesten-6-ol-3-one by cholesterol oxidase, and the observed differences in the rate of formation of the 6-hydroxy-ketosteroid by the enzymes of different bacterial sources are also discussed.

Extracellular 3β-hydroxysteroid oxidase of Mycobacterium vaccae VKM Ac-1815D

“…Enzyme molecular weight (62 ± 4 kDa), properties and multifunctional action corresponded to ChO from the relative organisms, rather than short-chain dehydrogenases [9][10][11].…”

Two moles of O2 consumption and one mole of H2O2 formation during cholesterol peroxidation with cholesterol oxidase from Pseudomonas sp. strain ST-200

“…The spontaneous conversion of 6β-hydroperoxycholest-4-en-3-one is reported for products with Pfl, although it has been proposed that 6ζ-hydroxycholest-4-en-3-one is produced from cholesterol by mono-oxygenation with Pse [15]. We conclude that the oxidase produces 6β-hydroperoxycholest-4-en-3-one initially and that other oxysteroids are produced from the initial product.…”

“…The production of cholest-4-ene-3,6-dione is rapid compared with that of 6β-hydroperoxycholest-4-en-3-one. Although 6β-hydroperoxycholest-4-en-3-one is spontaneously converted to several steroids including cholest-4-ene-3,6-dione [15], some unidentified biological process might be involved in the conversion. In addition, ST-200 does not grow on cholesterol or 6β-hydroperoxycholest-4-en-3-one, suggesting that cholesterol is not a natural substrate for this enzyme.…”

Control with Organic Solvents of Efficiency of Persolvent Cholesterol Fermentation byPseudomonassp. Strain ST-200

“…23) In contrary, it is also reported that Comamonas testosteroni has a L1 5 -3-ketosteroid isomerase distinctive from 3f3-hydroxysteroid oxidase. 24 ) A preliminary observation showed that the organism had a 3f3-hydroxysteroid oxidase capable of converting cholesterol mainly to C5EO.…”