title: Domain Characterization of Cyclosporin Regio-Specific Hydroxylases in Rare Actinomycetes.
abstract: Cytochrome P450 hydroxylase (CYP) in actinomycetes plays an important role in the biosynthesis and bioconversion of various secondary metabolites. Two unique CYPs named CYP-sb21 and CYP-pa1, which were identified from Sebekia benihana and Pseudonocardia autotrophica, respectively, were proven to transfer a hydroxyl group at the 4(th) or 9(th) N-methyl leucine position of immunosuppressive agent cyclosporin A (CsA). Interestingly, these two homologous CYPs showed different CsA regio-selectivities. CYP-sb21 exhibited preferential hydroxylation activity at the 4(th) position over the 9(th) position, whereas CYP-pa1 showed the opposite preference. To narrow down the CYP domain critical for CsA regio-selectivity, each CYP was divided into four domains, and each domain was swapped with its counterpart from the other CYP. A total of 18 hybrid CYPs were then individually tested for CsA regioselectivity. Although most of the hybrid CYPs failed to exhibit a significant change in regioselectivity in the context of CsA hydroxylation, hybrid CYP-pa1 swapped with the second domain of CYP-sb21 showed a higher preference for the 9th position. Moreover, hybrid CYPsb21 containing seven amino acids from the 2nd domain of CYP-pa1 showed higher preference for the 4(th) position. These results imply that the 2nd domain of CsA-specific CYP plays a critical role in CsA regio-selectivity, thereby setting the stage for biotechnological application of CsA regio-selective hydroxylation.
authors.affiliation: Department of Biological Engineering, Inha University, Incheon 402-751, Republic of Korea.
authors.forename: Min-Woo
authors.initials: MW
authors.lastname: Woo
authors.affiliation: Department of Biological Engineering, Inha University, Incheon 402-751, Republic of Korea.
authors.forename: Bo-Ram
authors.initials: BR
authors.lastname: Lee
authors.affiliation: Department of Biological Engineering, Inha University, Incheon 402-751, Republic of Korea.
authors.forename: Hee-Ju
authors.initials: HJ
authors.lastname: Nah
authors.affiliation: Department of Biological Engineering, Inha University, Incheon 402-751, Republic of Korea.
authors.forename: Si-Sun
authors.initials: SS
authors.lastname: Choi
authors.affiliation: Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong 266101, P.R. China.
authors.forename: Shengying
authors.initials: S
authors.lastname: Li
authors.affiliation: Department of Biological Engineering, Inha University, Incheon 402-751, Republic of Korea.
authors.forename: Eung-Soo
authors.initials: ES
authors.lastname: Kim
mesh_terms: D039903:Actinobacteria / Q000201:enzymology* / Q000235:genetics; D016572:Cyclosporine / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
chemical_list: D016572:Cyclosporine; D006899:Mixed Function Oxygenases
keywords: Actinomycetes; Cyclosporin A; Cytochrome P450; Regio-selectivity