Unnatural activities and mechanistic insights of cytochrome P450 PikC gained from site-specific mutagenesis by non-canonical amino acids

Yunjun Pan, Guobang Li, Ruxin Liu, Jiawei Guo, Yunjie Liu, Mingyu Liu, Xingwang Zhang, Luping Chi, Kangwei Xu, Ruibo Wu, Yuzhong Zhang, Yuezhong Li, Xiang Gao () and Shengying Li ()
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Yunjun Pan: Shandong University
Guobang Li: Shandong University
Ruxin Liu: Shandong University
Jiawei Guo: Shandong University
Yunjie Liu: Shandong University
Mingyu Liu: Shandong University
Xingwang Zhang: Shandong University
Luping Chi: Shandong University
Kangwei Xu: Sun Yat-sen University
Ruibo Wu: Sun Yat-sen University
Yuzhong Zhang: Shandong University
Yuezhong Li: Shandong University
Xiang Gao: Shandong University
Shengying Li: Shandong University

Nature Communications, 2023, vol. 14, issue 1, 1-14

Abstract: 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.

Date: 2023
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DOI: 10.1038/s41467-023-37288-0

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