Genetic modification of the Streptomyces cholesterol oxidase gene for expression in Escherichia coli and development of promoter-probe vectors for use in enteric bacteria

Nomura, Nobuhiko; Choi, Kwang Pil; Yamashita, Mitsuo; Yamamoto, Hiroko; Murooka, Yoshikatsu

doi:10.1016/0922-338x(95)91253-2

Cited by 34 publications

(20 citation statements)

References 32 publications

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“…The wild-type Streptomyces SA-COO CO gene was amplified from the pCO117 plasmid (Nomura et al, 1995) by polymerase chain reaction (PCR) using the forward and reverse primers 5 0 -GACTTCATGGCCACTGCACAACAG-CATCTG-3 0 and 3 0 -GCAGTGCCGCAGCGTGGTGGTGG-TGGTGGTGATTCGAACTGA-5 0 , respectively. The reverse primer included codons for six histidine residues.…”

Section: Cloningmentioning

confidence: 99%

“…The nonhistidine-tagged version of the protein (CO) was used for structure determination of the dithionite-treated crystals. Competent E. coli BL21 (DE3) pLysS cells were transformed with the pCO117 plasmid (Nomura et al, 1995) and transformants selected from LB agar plates supplemented with 50 mg ml À1 ampicillin and 34 mg ml À1 chloramphenicol. 50 ml LB containing the above antibiotics was inoculated with a single colony from the transformation and grown overnight at 310 K. This starter was used to inoculate 2 l of 2ÂYT medium also containing the above antibiotics.…”

Section: Expression and Purificationmentioning

confidence: 99%

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High-resolution structures of cholesterol oxidase in the reduced state provide insights into redox stabilization

Golden

Karton

Vrielink

2014

Acta Cryst D Biol Crystallogr

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Cholesterol oxidase (CO) is a flavoenzyme that catalyzes the oxidation and isomerization of cholesterol to cholest-4-en-3-one. The reductive half reaction occurs via a hydride transfer from the substrate to the FAD cofactor. The structures of CO reduced with dithionite under aerobic conditions and in the presence of the substrate 2-propanol under both aerobic and anaerobic conditions are presented. The 1.32 Å resolution structure of the dithionite-reduced enzyme reveals a sulfite molecule covalently bound to the FAD cofactor. The isoalloxazine ring system displays a bent structure relative to that of the oxidized enzyme, and alternate conformations of a triad of aromatic residues near to the cofactor are evident. A 1.12 Å resolution anaerobically trapped reduced enzyme structure in the presence of 2-propanol does not show a similar bending of the flavin ring system, but does show alternate conformations of the aromatic triad. Additionally, a significant difference electron-density peak is observed within a covalent-bond distance of N5 of the flavin moiety, suggesting that a hydride-transfer event has occurred as a result of substrate oxidation trapping the flavin in the electron-rich reduced state. The hydride transfer generates a tetrahedral geometry about the flavin N5 atom. High-level density-functional theory calculations were performed to correlate the crystallographic findings with the energetics of this unusual arrangement of the flavin moiety. These calculations suggest that strong hydrogen-bond interactions between Gly120 and the flavin N5 centre may play an important role in these structural features.

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Section: Cloningmentioning

confidence: 99%

Section: Expression and Purificationmentioning

confidence: 99%

High-resolution structures of cholesterol oxidase in the reduced state provide insights into redox stabilization

Golden

Karton

Vrielink

2014

Acta Cryst D Biol Crystallogr

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“…The secretory overproduction of Streptomyces cholesterol oxidase (ChoA S ) in a Streptomyces host-vector system was demonstrated (Molnár et al, 1991). Recently, the expression of the choA gene in Escherichia coli by genetic modification was also reported (Nomura et al, 1995). The crystal structure of cholesterol oxidase (ChoA B ) from Brevibacterium sterolicum was determined and refined at 1.8 Å resolution, which provided a complete structural description of the enzyme (Vrielink et al, 1991).…”

Section: Introductionmentioning

confidence: 99%

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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“…The wild-type S. SA-COO cholesterol oxidase gene was amplified from pCO117 [27] and cloned into the Nco1 and HindIII restriction sites of pET28a-His6-MBP-TEV-AEW. The forward and reverse primers are 5 0 -gactccatggccactgcacaacagcatctg-3 0 and 3 0 -gcagtgccgca gcgtggtggtggtggtggtgattcgaactga-5 0 , respectively.…”

Section: Cloningmentioning

confidence: 99%

Production and characterization of recombinant perdeuterated cholesterol oxidase

Golden

Attwood

Duff

et al. 2015

Analytical Biochemistry

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Genetic modification of the Streptomyces cholesterol oxidase gene for expression in Escherichia coli and development of promoter-probe vectors for use in enteric bacteria

Cited by 34 publications

References 32 publications

High-resolution structures of cholesterol oxidase in the reduced state provide insights into redox stabilization

High-resolution structures of cholesterol oxidase in the reduced state provide insights into redox stabilization

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

Production and characterization of recombinant perdeuterated cholesterol oxidase

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