Cloning and expression of a Streptomyces cholesterol oxidase gene in Streptomyces lividans with plasmid pIJ702

Murooka, Y; Ishizaki, Takayuki; Nimi, Osamu; Maekawa, N

doi:10.1128/aem.52.6.1382-1385.1986

Cited by 75 publications

(15 citation statements)

References 20 publications

(17 reference statements)

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“…Unidentified compound V was detectable in reaction mixtures containing 4-cholesten-3one and protein extracts from Streptomyces strains, regardless of their plasmid content. Compound IV, on the other hand, was missing when a protein extract from Streptomyces tividans (pUC702) (the strain carrying a shuttle cloning vector; Molnar efa/., 1991) was employed, but appeared when protein extracts from S. tividans (pCOl) (Murooka et at., 1986) COO were utilized to degrade cholesterol or 4-cholesten-3-one as substrates, respectively. Under the conditions employed, compound IV appeared to be the only stable and relatively abundant cholesterol metabolite accumulating exclusively in reaction mixtures containing protein extracts of Streptomyoes sp.…”

Section: Detection Of a New Ohotesterot Biodegradation Product By Hplcmentioning

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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Section: Detection Of a New Ohotesterot Biodegradation Product By Hplcmentioning

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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“…We previously cloned and sequenced the gene for cholesterol oxidase (choA) from Streptomyces sp. SA-COO (Murooka et al, 1986;Ishizaki et al, 1989). The secretory overproduction of Streptomyces cholesterol oxidase (ChoA S ) in a Streptomyces host-vector system was demonstrated (Molnár et al, 1991).…”

Section: Introductionmentioning

confidence: 98%

Separation of the two reactions, oxidation and isomerization, catalyzed by Streptomyces cholesterol oxidase

Yamashita¹,

Toyama²,

Ono³

et al. 1998

Protein Engineering Design and Selection

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Site-directed mutagenesis was used to identify key amino acid residues of the cholesterol oxidase from Streptomyces sp., which catalyzes the oxidation of cholesterol and the isomerization of 5-cholesten-3-one. Eight mutant enzymes were constructed and the following amino acid substitutions were identified: N318A, N318H, E356A, E356D, H441A, H441N, N480A and N480Q. Mutants N318A and N318H retained both oxidation and isomerization activities. The mutant E356D retained oxidation but not isomerization activity. On the other hand, mutants N480A and N480Q showed no oxidation activity but retained their isomerization activities. The two catalytic reactions, oxidation and isomerization, in cholesterol oxidase were thus successfully separated. When the H441A or H441N mutation was introduced, both the oxidase and isomerase activities were completely lost. The H441, E356 and N480 residues thus appear to participate in the catalysis of cholesterol oxidase, whereas N318 does not. An analysis of the products of these mutant enzymes suggested that the previously proposed 6-hydroxylation reaction by cholesterol oxidase is actually autooxidation from 5-cholesten-3-one. Kinetic studies of the purified wild-type and mutant enzymes showed that the k(cat)/Km values for oxidation in E356D and for isomerization in N480A increased six- and threefold, respectively, over those in the wild-type. These mutational effects and the reaction mechanisms are discussed in terms of the three-dimensional structure of the enzyme constructed on the basis of homology modeling.

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“…Genetically engineered Streptomyces species may someday play important roles in the industrial production of useful polypeptides (19,21) and in the decomposition and bioconversion of organic materials (7,20). There currently is a high level of interest in the use of Streptomyces species in cloning experiments.…”

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confidence: 99%

Transfer of conjugative plasmids and mobilization of a nonconjugative plasmid between Streptomyces strains on agar and in soil

Rafii

Crawford

1988

Appl Environ Microbiol

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The conjugative plasmid pUJlOl and its conjugative nondeletion derivatives pIJ303 and pU211 were tested for their transferability between strains of Streptomyces on laboratory media and in the soil environment. Their roles in the mobilization of the cloning vector plasmid pIJ702, a nonconjugative deletion derivative of pLJ101, were also examined. Biparental and triparental crosses were performed on agar slants and in sterile soil between the plasmid donor Streptomyces lividans and several recipient Streptomyces strains previously isolated from soil. Conjugative plasmids were transferred to seven recipients in slant crosses and to three recipients in soil. Plasmids isolated from recipients showed restriction fragment patterns identical to that of the original plasmid in S. lividans. Plasmid pIJ303 was transferred less frequently in soil than on slants, and the frequency of transfer was higher at 30°C than at the other temperatures examined. Transconjugant Streptomyces strains differed in their ability to maintain pIJ303. The nonconjugative plasmid pIJ702 was mobilized on agar slants into S. coelicolor 2708, which already contains a self-transmissible plasmid. Plasmid pU702 was also mobilized into S. flavovirens, Streptomyces sp. strain 87A, and S. parvulus on slants and in sterile soil after triparental crosses with two donors, one containing pIJ702 and the other containing either pUJlOl or p1J211. The presence of a conjugative plasmid donor was required for the transfer of pIJ702 to S. parvulus 1234, S. flavovirens 28, and Streptomyces sp. strain 87A. Plasmid pIJ702 was always transferred in its normal, autonomous form. Chromosomal recombination also occurred in transconjugants after the transfer of pIJ702. This is the first report of gene transfer between Streptomyces strains in soil. * Corresponding author. t Paper no. 87517 of the Idaho Agricultural Experiment Station.

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Cloning and expression of a Streptomyces cholesterol oxidase gene in Streptomyces lividans with plasmid pIJ702

Cited by 75 publications

References 20 publications

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

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

Separation of the two reactions, oxidation and isomerization, catalyzed by Streptomyces cholesterol oxidase

Transfer of conjugative plasmids and mobilization of a nonconjugative plasmid between Streptomyces strains on agar and in soil

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