The substrate specificity and stereochemistry, reversibility and inhibition of the 3-oxo steroid Δ4-Δ5-isomerase component of cholesterol oxidase

Smith, Andrew G.; Brooks, C. J. W.

doi:10.1042/bj1670121

Cited by 66 publications

(12 citation statements)

References 38 publications

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“…Moreover, all characterized examples of actinomycetes that catabolize 3β-hydroxysterols for energy use, e.g., Rhodococcus (44,45) and Streptomyces (46), secrete the sterol oxidizing enzyme. Lastly, in contrast to the Mtb Rv1106c gene product, 3-hydroxy sterol oxidases known to be involved in primary metabolism are more specific for cholesterol than dehydroepiandrosterone or pregnenolone (47)(48)(49).…”

Section: Discussionmentioning

confidence: 99%

Rv1106c from Mycobacterium tuberculosis Is a 3β-Hydroxysteroid Dehydrogenase

et al. 2007

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New approaches are required to combat M. tuberculosis (Mtb), especially the multi and extremely drug resistant and organisms (MDR-TB and XDR-TB). There are many reports that mycobacteria oxidize 3β-hydroxysterols to 3-ketosteroids, but the enzyme(s) responsible for this activity have not been identified in mycobacterial species. In this work, the Rv1106c gene that is annotated as a 3β-hydroxysteroid dehydrogenase in Mtb has been cloned and heterologously expressed. The purified enzyme was kinetically characterized and found to have a pH optimum between 8.5 and 9.5. The enzyme, which is a member of the short chain dehydrogenase superfamily, uses NAD + as a cofactor and oxidizes cholesterol, pregnenolone and dehydroepiandrosterone to their respective 3-keto-4-ene products. The enzyme forms a ternary complex with NAD + binding before the sterol. The enzyme shows no substrate preference for dehydroepiandrosterone versus pregnenolone with second-order rate constants (k cat /K m ) of 3.2 ± 0.4 μM −1 min −1 and 3.9 ± 0.9 μM −1 min −1 , respectively at pH 8.5, 150 mM NaCl, 30 mM MgCl 2 , and saturating NAD + . Trilostane is a competitive inhibitor of dehydroepiandrosterone with a K i of 197 ± 8 |μM. The expression of the 3β-hydroxysteroid dehydrogenase in Mtb is intracellular. Disruption of the 3β-hydroxysteroid dehydrogenase gene in Mtb abrogates mycobacterial cholesterol oxidation activity. These data are consistent with Rv1106c being the gene responsible for 3β-hydroxysterol oxidation in Mtb.Tuberculosis is an opportunistic infection caused by Mycobacterium tuberculosis (Mtb 1 ) in individuals with HIV-AIDS that is estimated to infect 30% of the world's population (1,2). The World Health Organization estimates that 2 million people die every year from tuberculosis. Drug resistance to front-line Mtb drugs rifampicin and isoniazid has emerged (3,4) and additional resistance to second line drugs is emerging (5,6). It is clear that new approaches are required to combat these multi drug-resistant and extreme (or extensively) drugresistant organisms (7-9).The complete genome sequences of microorganisms are a rich source for mining new drug targets. However, oftentimes, biochemical functions have been assigned to genes purely on the basis of their sequence homology to gene products which are themselves poorly † Financial support from the National Institutes of Health (AI065251 (N.S.S.), AI065997 (I.S.), RR021008, (N.S.S.) and NIAID Contract# HHSN266200400091C, (Colorado State University) and the National Science Foundation (CHE0131146 for NMR spectrometers) is gratefully acknowledged. *corresponding author: Address: Stony Brook University, Stony Brook, New York 11794-3400, Phone: (631) Fax: (631) 632-5731 Nicole.Sampson@StonyBrook.edu. 1 Mtb: Mycobacterium tuberculosis; MDR-TB: multi-drug resistant Mycobacterium tuberculosis; XDR-TB: extremely-drug resistant Mycobacterium tuberculosis; DHEA: dehydroepiandrosterone; rH 6 3BHSD: recombinant 3β-hydroxysteroid dehydrogenase with an Nterminal six histidine tag; rSBHSD...

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Section: Discussionmentioning

confidence: 99%

Rv1106c from Mycobacterium tuberculosis Is a 3β-Hydroxysteroid Dehydrogenase

et al. 2007

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“…sitosterol), has little effect on substrate specificity when the steroids are monolayers [37] or in dioleoylphosphatidylcholine ⁄ sterol unilamellar vesicles [29]. This lack of specificity is in distinct contrast to substrate specificity studies in detergent micelles or with propan-2-ol co-solvent that demonstrates a specificity for cholesterol over sitosterol, androsten-3b-ol and related steroid structures (see the third review of this miniseries) [38,39]. Two types of model membrane have been used to study ChOx specificity for cholesterol in different mem- branes, namely unilamellar vesicles and monolayers.…”

Section: Enzyme Substrate Specificitymentioning

confidence: 99%

Cholesterol oxidase: physiological functions

Kreit¹,

Sampson²

2009

The FEBS Journal

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An important aspect of catalysis performed by cholesterol oxidase (3β‐hydroxysteroid oxidase) concerns the nature of its association with the lipid bilayer that contains the sterol substrate. Efficient catalytic turnover is affected by the association of the protein with the membrane as well as the solubility of the substrate in the lipid bilayer. In this review, the binding of cholesterol oxidase to the lipid bilayer, its turnover of substrates presented in different physical environments, and how these conditions affect substrate specificity, are discussed. The physiological functions of the enzyme in bacterial metabolism, pathogenesis and macrolide biosynthesis are reviewed in this context.

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“…Cholesterol oxidase (cholesterol: O2 oxidoreductase, EC 1.1.3.6. ), a bifunctional enzyme, catalyses the oxidation of cholesterol to the temporai7 intermediate 5cholesten-3-one with the reduction of molecular oxygen to hydrogen peroxide (Stadtman etat., 1954;Uwajima ef at., 1974), and the isomehzation of the A^-bond (Smith and Brooks, 1977), via a mechanism analogous to that of the ii^-3-ketosteroid isomerase from Pseudomonas testosterini (Batzold ef at., 1976;Kuliopulos ef at.. 1987) to produce 4-cholesten-3-one as a final product. Cholesterol oxidases were isolated and characterized from numerous bacterial sources (Noma and Nakayama, 1976), and most of them were shown to contain one mole of tightly bound flavin adenin dinucleotide (FAD) per mole of protein as a prosthetic group (Uwajima efa/., 1974;Kameiefa/., 1978 Kenney ef at., 1979), although some of them, for example the enzyme from Nocardia erythropotis, lack this cofactor (Richmond, 1973).…”

Section: Introductionmentioning

confidence: 99%

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

Molnár

Hayashi

Choi

et al. 1993

Molecular Microbiology

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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.

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The substrate specificity and stereochemistry, reversibility and inhibition of the 3-oxo steroid Δ4-Δ5-isomerase component of cholesterol oxidase

Cited by 66 publications

References 38 publications

Rv1106c from Mycobacterium tuberculosis Is a 3β-Hydroxysteroid Dehydrogenase

Rv1106c from Mycobacterium tuberculosis Is a 3β-Hydroxysteroid Dehydrogenase

Cholesterol oxidase: physiological functions

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

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