title: Integrating cell-free biosyntheses of heme prosthetic group and apoenzyme for the synthesis of functional P450 monooxygenase.
abstract: Harnessing the isolated protein synthesis machinery, cell-free protein synthesis reproduces the cellular process of decoding genetic information in artificially controlled environments. More often than not, however, generation of functional proteins requires more than simple translation of genetic sequences. For instance, many of the industrially important enzymes require non-protein prosthetic groups for biological activity. Herein, we report the complete cell-free biogenesis of a heme prosthetic group and its integration with concurrent apoenzyme synthesis for the production of functional P450 monooxygenase. Step reactions required for the syntheses of apoenzyme and the prosthetic group have been designed so that these two separate pathways take place in the same reaction mixture, being insulated from each other. Combined pathways for the synthesis of functional P450 monooxygenase were then further integrated with in situ assay reactions to enable real-time measurement of enzymatic activity during its synthesis.
authors.affiliation: Department of Fine Chemical Engineering and Applied Chemistry, Chungnam National University, Daejeon 305-764, Korea.
authors.forename: Yong-Chan
authors.initials: YC
authors.lastname: Kwon
authors.forename: In-Seok
authors.initials: IS
authors.lastname: Oh
authors.forename: Nahum
authors.initials: N
authors.lastname: Lee
authors.forename: Kyung-Ho
authors.initials: KH
authors.lastname: Lee
authors.forename: Yeo Joon
authors.initials: YJ
authors.lastname: Yoon
authors.forename: Eun Yeol
authors.initials: EY
authors.lastname: Lee
authors.forename: Byung-Gee
authors.initials: BG
authors.lastname: Kim
authors.forename: Dong-Myung
authors.initials: DM
authors.lastname: Kim
mesh_terms: D002474:Cell-Free System; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000378:metabolism; D006418:Heme / Q000096:biosynthesis* / Q000378:metabolism
chemical_list: D006418:Heme; D003577:Cytochrome P-450 Enzyme System