Molecular basis of regio- and stereo-specificity in biosynthesis of bacterial heterodimeric diketopiperazines

Chenghai Sun,Wenya Tian,Xudong Qu,Zixin Deng,Zhenyao Luo,Haidong Peng,Xinying Jia,Zhi Lin,Bostjan Kobe,Wenlu Zhang

doi:10.1038/s41467-020-20022-5

Chenghai Sun, Wenya Tian + Show 8 more

Open Access

https://doi.org/10.1038/s41467-020-20022-5

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Abstract

Bacterial heterodimeric tryptophan-containing diketopiperazines (HTDKPs) are a growing family of bioactive natural products. They are challenging to prepare by chemical routes due to the polycyclic and densely functionalized backbone. Through functional characterization and investigation, we herein identify a family of three related HTDKP-forming cytochrome P450s (NasbB, NasS1868 and NasF5053) and reveal four critical residues (Qln65, Ala86, Ser284 and Val288) that control their regio- and stereo-selectivity to generate diverse dimeric DKP frameworks. Engineering these residues can alter the specificities of the enzymes to produce diverse frameworks. Determining the crystal structures (1.70–1.47 Å) of NasF5053 (ligand-free and substrate-bound NasF5053 and its Q65I-A86G and S284A-V288A mutants) and molecular dynamics simulation finally elucidate the specificity-conferring mechanism of these residues. Our results provide a clear molecular and mechanistic basis into this family of HTDKP-forming P450s, laying a solid foundation for rapid access to the molecular diversity of HTDKP frameworks through rational engineering of the P450s.

Highlights

Bacterial heterodimeric tryptophan-containing diketopiperazines (HTDKPs) are a growing family of bioactive natural products
To develop efficient biocatalytic approaches, we recently investigated the biosynthesis of naseseazine C (NAS-C) and identified a key diketopiperazine (DKP) forming P450 enzyme (NascB)[6]
The P450 NascB (CYP nomenclature: CYP1190B2) encoded in locus-1 was responsible for the biosynthesis of NAS-C6

Summary

Introduction

Bacterial heterodimeric tryptophan-containing diketopiperazines (HTDKPs) are a growing family of bioactive natural products. HTDKP-forming P450s have relaxed regio-specificity and stereo-specificity and can generate products with different frameworks, e.g., AspB is able to convert cyclo-L-Trp-L-Pro (cWL-PL) into NAS-C (type III) and iso-NASB (type IV) accompanying the major product ASP-A (type V)[11] This property of co-generation of different types of HTDKPs suggests these P450s have a regulatory mechanism in controlling different regio-specificities and stereo-specificities and presents a great potential for further improving catalytic efficiency, altering specificity and even creating diverse frameworks by rational protein engineering. Such endeavors are reliant on understanding the molecular basis of HTDKPs-producing P450catalyzed reactions, which currently remains elusive. Based on our structural characterization, molecular dynamics and mutagenesisvalidation of the residues involved, we elucidate how the regioconfiguration and stereo-configuration in forming bonds is finely tuned in these P450s

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