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Unnatural activities and mechanistic insights of cytochrome P450 PikC gained from site-specific mutagenesis by non-canonical amino acids.
Pan, Yunjun; Li, Guobang; Liu, Ruxin; Guo, Jiawei; Liu, Yunjie; Liu, Mingyu; Zhang, Xingwang; Chi, Luping; Xu, Kangwei; Wu, Ruibo; Zhang, Yuzhong; Li, Yuezhong; Gao, Xiang; Li, Shengying.
Afiliação
  • Pan Y; State Key Laboratory of Microbial Technology, Shandong University, Qingdao, Shandong, 266237, China.
  • Li G; State Key Laboratory of Microbial Technology, Shandong University, Qingdao, Shandong, 266237, China.
  • Liu R; State Key Laboratory of Microbial Technology, Shandong University, Qingdao, Shandong, 266237, China.
  • Guo J; State Key Laboratory of Microbial Technology, Shandong University, Qingdao, Shandong, 266237, China.
  • Liu Y; State Key Laboratory of Microbial Technology, Shandong University, Qingdao, Shandong, 266237, China.
  • Liu M; State Key Laboratory of Microbial Technology, Shandong University, Qingdao, Shandong, 266237, China.
  • Zhang X; State Key Laboratory of Microbial Technology, Shandong University, Qingdao, Shandong, 266237, China.
  • Chi L; Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao, Shandong, 266237, China.
  • Xu K; State Key Laboratory of Microbial Technology, Shandong University, Qingdao, Shandong, 266237, China.
  • Wu R; School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, 510006, China.
  • Zhang Y; School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, 510006, China.
  • Li Y; State Key Laboratory of Microbial Technology, Shandong University, Qingdao, Shandong, 266237, China.
  • Gao X; Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao, Shandong, 266237, China.
  • Li S; State Key Laboratory of Microbial Technology, Shandong University, Qingdao, Shandong, 266237, China.
Nat Commun ; 14(1): 1669, 2023 03 25.
Article em En | MEDLINE | ID: mdl-36966128
ABSTRACT
Cytochrome P450 enzymes play important roles in the biosynthesis of macrolide antibiotics by mediating a vast variety of regio- and stereoselective oxidative modifications, thus improving their chemical diversity, biological activities, and pharmaceutical properties. Tremendous efforts have been made on engineering the reactivity and selectivity of these useful biocatalysts. However, the 20 proteinogenic amino acids cannot always satisfy the requirement of site-directed/random mutagenesis and rational protein design of P450 enzymes. To address this issue, herein, we practice the semi-rational non-canonical amino acid mutagenesis for the pikromycin biosynthetic P450 enzyme PikC, which recognizes its native macrolide substrates with a 12- or 14-membered ring macrolactone linked to a deoxyamino sugar through a unique sugar-anchoring mechanism. Based on a semi-rationally designed substrate binding strategy, non-canonical amino acid mutagenesis at the His238 position enables the unnatural activities of several PikC mutants towards the macrolactone precursors without any sugar appendix. With the aglycone hydroxylating activities, the pikromycin biosynthetic pathway is rewired by the representative mutant PikCH238pAcF carrying a p-acetylphenylalanine residue at the His238 position and a promiscuous glycosyltransferase. Moreover, structural analysis of substrate-free and three different enzyme-substrate complexes of PikCH238pAcF provides significant mechanistic insights into the substrate binding and catalytic selectivity of this paradigm biosynthetic P450 enzyme.
Assuntos

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Sistema Enzimático do Citocromo P-450 / Aminoácidos Idioma: En Revista: Nat Commun Ano de publicação: 2023 Tipo de documento: Article

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Sistema Enzimático do Citocromo P-450 / Aminoácidos Idioma: En Revista: Nat Commun Ano de publicação: 2023 Tipo de documento: Article