Engineering bacterial cytochrome P450 (P450) BM3 into a prototype with human P450 enzyme activity using indigo formation

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dc.contributor.authorS H Park-
dc.contributor.authorD H Kim-
dc.contributor.authorDoo Il Kim-
dc.contributor.authorD H Kim-
dc.contributor.authorHeung Chae Jung-
dc.contributor.authorJae Gu Pan-
dc.contributor.authorT Ahn-
dc.contributor.authorD Kim-
dc.contributor.authorC H Yun-
dc.date.accessioned2017-04-19T09:18:32Z-
dc.date.available2017-04-19T09:18:32Z-
dc.date.issued2010-
dc.identifier.issn0090-9556-
dc.identifier.uri10.1124/dmd.109.030759ko
dc.identifier.urihttps://oak.kribb.re.kr/handle/201005/9511-
dc.description.abstractHuman cytochrome P450 (P450) enzymes metabolize a variety of endogenous and xenobiotic compounds, including steroids, drugs, and environmental chemicals. In this study, we examine the possibility that bacterial P450 BM3 (CYP102A1) mutants with indole oxidation activity have the catalytic activities of human P450 enzymes. Errorprone polymerase chain reaction was carried out on the heme domain-coding region of the wild-type gene to generate a CYP102A1 DNA library. The library was transformed into Escherichia coli for expression of the P450 mutants. A colorimetric colony-based method was adopted for primary screening of the mutants. When the P450 activities were measured at the whole-cell level, some of the blue colonies, but not the white colonies, possessed apparent oxidation activity toward coumarin and 7-ethoxycoumarin, which are typical human P450 substrates that produce fluorescent products. Coumarin is oxidized by the CYP102A1 mutants to produce two metabolites, 7-hydroxycoumarin and 3-hydroxycoumarin. In addition, 7-ethoxycoumarin is simultaneously oxidized to 7-hydroxycoumarin by O-deethylation reaction and to 3-hydroxy,7-ethoxycoumarin by 3-hydroxylation reactions. Highly active mutants are also able to metabolize several other human P450 substrates, including phenacetin, ethoxyresorufin, and chlorzoxazone. These results indicate that indigo formation provides a simple assay for identifying CYP102A1 mutants with a greater potential for human P450 activity. Furthermore, our computational findings suggest a correlation between the stabilization of the binding site and the catalytic efficiency of CYP102A1 mutants toward coumarin: the more stable the structure in the binding site, the lower the energy barrier and the higher the catalytic efficiency.-
dc.publisherAmer Soc Pharmacology Experimental Therapeutics-
dc.titleEngineering bacterial cytochrome P450 (P450) BM3 into a prototype with human P450 enzyme activity using indigo formation-
dc.title.alternativeEngineering bacterial cytochrome P450 (P450) BM3 into a prototype with human P450 enzyme activity using indigo formation-
dc.typeArticle-
dc.citation.titleDrug Metabolism and Disposition-
dc.citation.number5-
dc.citation.endPage739-
dc.citation.startPage732-
dc.citation.volume38-
dc.contributor.affiliatedAuthorDoo Il Kim-
dc.contributor.affiliatedAuthorHeung Chae Jung-
dc.contributor.affiliatedAuthorJae Gu Pan-
dc.contributor.alternativeName박선하-
dc.contributor.alternativeName김동현-
dc.contributor.alternativeName김두일-
dc.contributor.alternativeName김대환-
dc.contributor.alternativeName정흥채-
dc.contributor.alternativeName반재구-
dc.contributor.alternativeName안태호-
dc.contributor.alternativeName김동학-
dc.contributor.alternativeName윤철호-
dc.identifier.bibliographicCitationDrug Metabolism and Disposition, vol. 38, no. 5, pp. 732-739-
dc.identifier.doi10.1124/dmd.109.030759-
dc.description.journalClassY-
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Division of Bio Technology Innovation > SME Support Center > 1. Journal Articles
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