8GUE

Crystal Structure of narbomycin-bound cytochrome P450 PikC with the unnatural amino acid p-Acetyl-L-Phenylalanine incorporated at position 238

Summary for 8GUE

• Entry DOI: 10.2210/pdb8gue/pdb
• Descriptor: Cytochrome P450 monooxygenase PikC, PROTOPORPHYRIN IX CONTAINING FE, NARBOMYCIN, ... (8 entities in total)
• Functional Keywords: oxidoreductase, narbomycin-bound, pikc, unnatural amino acid
• Biological source: Streptomyces venezuelae
• Total number of polymer chains: 2
• Total formula weight: 98206.74

Authors

Li, G.B.,Pan, Y.J.,Li, S.Y.,Gao, X. (deposition date: 2022-09-11, release date: 2023-02-15, Last modification date: 2023-11-29)

Primary citation

Pan, Y.,Li, G.,Liu, R.,Guo, J.,Liu, Y.,Liu, M.,Zhang, X.,Chi, L.,Xu, K.,Wu, R.,Zhang, Y.,Li, Y.,Gao, X.,Li, S.
Unnatural activities and mechanistic insights of cytochrome P450 PikC gained from site-specific mutagenesis by non-canonical amino acids.
Nat Commun, 14:1669-1669, 2023

PubMed 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 PikC 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 PikC provides significant mechanistic insights into the substrate binding and catalytic selectivity of this paradigm biosynthetic P450 enzyme.

PubMed: 36966128
DOI: 10.1038/s41467-023-37288-0

Experimental method

X-RAY DIFFRACTION (1.9 Å)