Properties of Crystalline 3β-Hydroxysteroid Oxidase of Brevibacterium sterolicum

UWAJIMA, Takayuki; YAGI, Hitoshi; Terada, Osamu

doi:10.1271/bbb1961.38.1149

Cited by 64 publications

(40 citation statements)

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“…In addition, FMN-complexed proteins act as intermediaries in the transfer of electrons in biological oxidation-reduction reactions. Interestingly, in some bacteria, flavin adenine dinucleotide (FAD) and FMN act as cholesterol oxidases (9,33), and other studies have shown that cholesterol oxidases from numerous bacteria tightly bind the flavin prosthetic group (11,20,21,37,56). In addition, the major facilitator superfamily (MFS) protein (spot 3), which was downregulated in the BA9 mutant in the presence of cholesterol with bile, is the largest family of secondary transport carriers found in living organisms, including bacteria.…”

Section: Resultsmentioning

confidence: 99%

Genetic and Proteomic Analysis of Factors Affecting Serum Cholesterol Reduction by Lactobacillus acidophilus A4

Liu

Kim

Yun

et al. 2010

Appl Environ Microbiol

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This article identifies novel factors involved in cholesterol reduction by probiotic bacteria, which were identified using genetic and proteomic approaches. Approximately 600 Lactobacillus acidophilus A4 mutants were created by random mutagenesis. The cholesterol-reducing ability of each mutant was determined and verified using two different methods: the o-phthalaldehyde assay and gas chromatographic analysis (GC). Among screened mutants, strain BA9 showed a dramatically diminished ability to reduce cholesterol, as demonstrated by a 7.7% reduction rate, while the parent strain had a more than 50% reduction rate. The transposon insertion site was mapped using inverse PCR (I-PCR), and it was determined using bioinformatic methods that the deleted region contained the Streptococcus thermophilus catabolite control protein A gene (ccpA). In addition, we have shown using two-dimensional gel electrophoresis (2-DE) that several proteins, including a transcription regulator, FMN-binding protein, major facilitator superfamily permease, glycogen phosphorylase, the YknV protein, and fructose/tagatose bisphosphate aldolase, were strongly regulated by the ccpA gene. In addition, in vivo experiments investigating ccpA function were conducted with rats. Rats fed wild-type L. acidophilus A4 showed a greater than 20% reduction in total serum cholesterol, but rats fed BA9 mutant L. acidophilus showed only an approximately 10% reduction in cholesterol. These results provide important insights into the mechanism by which these lactic acid bacteria reduce cholesterol.

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Section: Resultsmentioning

confidence: 99%

Genetic and Proteomic Analysis of Factors Affecting Serum Cholesterol Reduction by Lactobacillus acidophilus A4

Liu

Kim

Yun

et al. 2010

Appl Environ Microbiol

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“…is a FAD-dependent enzyme that catalyzes the oxidation and isomerization of cholesterol into 4-cholesten-3-one (cholestenone) and yielding H 2 O 2 (Uwajima et al 1974). This enzyme is industrially important and widely used for the determination of cholesterol in food and blood serum by coupling with peroxidase (Allain et al 1974;Richmond 1992), and also in the production of starting material for the chemical synthesis of pharmaceutical steroids.…”

Section: Introductionmentioning

confidence: 99%

“…Cholesterol oxidase is produced by several microorganisms including Brevibacterium (Uwajima et al 1974), Streptomyces spp. (Tomioka et al 1976), Corynebacterium (Shirokano et al 1977), Arthrobacter (Liu et al 1983), Pseudomonas (Aono et al 1994), and Rhodococcus (Kreit et al 1994, Sojo et al 1997.…”

Section: Introductionmentioning

confidence: 99%

Coenzyme precursor-assisted expression of a cholesterol oxidase from Brevibacterium sp. in Escherichia coli

Wang

2007

Biotechnol Lett

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The gene (choB(b)), encoding cholesterol oxidase from Brevibacterium sp. CCTCC M201008, was cloned and sequenced by PCR (GenBank accession number: DQ345780). The gene consists of 1653 base pairs and encodes a protein of 551 amino acids. ChoB(b) exhibited a homology of 98% with cholesterol oxidase gene from Brevibacterium sterolicum ATCC 21387. The cholesterol oxidase gene, cloned in the vector pET-28a, was over-expressed in Escherichia coli BL21-CodonPlus (DE3)-RP grown at 23 degrees C in Luria-Bertani medium containing 50 microM riboflavin, the precursor of the FAD coenzyme of the enzyme. A maximum activity of 3.7 U/mg was obtained from cell free extract of E. coli BL21-CodonPlus (DE3)-RP harboring the pET-28a-choB(b).

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“…ATCC21387 extracellularly producing cholesterol oxidase and puri¢ed the enzyme to a crystalline form from the culture supernatant [1]. The crystalline cholesterol oxidase was a £avoprotein containing ¢rmly bound FAD as a prosthetic group [2]. The crystal structure analysis of the enzyme at 1.8 A î resolution made clear that it consisted of a FAD-binding domain and a steroid-binding domain [3].…”

Section: Introductionmentioning

confidence: 99%

Purification and properties of a new Brevibacterium sterolicum cholesterol oxidase produced by E. coli MM294/pnH10

Fujishiro

2002

FEMS Microbiology Letters

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A gene encoding a cholesterol oxidase from Brevibacterium sterolicum nov. sp. ATCC21387 was isolated by an expression cloning method and highly expressed by a recombinant strain Escherichia coli MM294/pnH10. The purified cholesterol oxidase was a typical flavoprotein with a molecular mass of 46.5 kDa, absorption peaks at 280, 360, and 450 nm. Optimum pH and temperature were found at pH 6.5 and 55 ‡C, respectively. The enzyme acted on 3L-hydroxysteroids such as cholesterol, pregnenolone, and L-sitosterol at high rates, but on dehydro-epi-androsterone to a lesser degree. The molecular and catalytic properties were different from those of cholesterol oxidase I, which was initially discovered in B. sterolicum nov. sp. ATCC21387. The new enzyme, designated cholesterol oxidase II, was distinguished by its high affinity toward cholesterol (K m = 30 WM).

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Properties of Crystalline 3β-Hydroxysteroid Oxidase of Brevibacterium sterolicum

Cited by 64 publications

References 0 publications

Genetic and Proteomic Analysis of Factors Affecting Serum Cholesterol Reduction by Lactobacillus acidophilus A4

Genetic and Proteomic Analysis of Factors Affecting Serum Cholesterol Reduction by Lactobacillus acidophilus A4

Coenzyme precursor-assisted expression of a cholesterol oxidase from Brevibacterium sp. in Escherichia coli

Purification and properties of a new Brevibacterium sterolicum cholesterol oxidase produced by E. coli MM294/pnH10

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