Volume 32, No 3, Mar 2022

ISSN: 1001-0602 
EISSN: 1748-7838 2018 
impact factor 17.848* 
(Clarivate Analytics, 2019)

Volume 32 Issue 3, March 2022: 302-314   |  Open Access

ORIGINAL ARTICLES

Crystal structure and catalytic mechanism of the MbnBC holoenzyme required for methanobactin biosynthesis

Chao Dou^1,† , Zhaolin Long^1,† , Shoujie Li^1,† , Dan Zhou^1,† , Ying Jin^1,† , Li Zhang^1,† , Xuan Zhang² , Yanhui Zheng¹ , Lin Li³ , Xiaofeng Zhu^1,4 , Zheng Liu⁵ , Siyu He¹ , Weizhu Yan¹ , Lulu Yang¹ , Jie Xiong¹ , Xianghui Fu¹ , Shiqian Qi¹ , Haiyan Ren¹ , She Chen³ , Lunzhi Dai¹ , Binju Wang² , Wei Cheng^1,*

¹Division of Respiratory and Critical Care Medicine, Respiratory Infection and Intervention Laboratory of Frontiers Science Center for Disease-related Molecular Network, State Key Laboratory of Biotherapy, West China Hospital of Sichuan University, Chengdu, Sichuan, China
²State Key Laboratory of Structural Chemistry of Solid Surface and Fujian Provincial Key Laboratory of Theoretical and Computational Chemistry, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian, China
³National Institute of Biological Sciences, NIBS, Beijing, China
⁴College of Life Science, Sichuan University, Chengdu, Sichuan, China
⁵Beijing National Laboratory for Molecular Sciences (BNLMS), Beijing Key Laboratory for Magnetoelectric Materials and Devices, College of Chemistry and Molecular Engineering, Peking University, Beijing, China
^†These authors contributed equally: Chao Dou, Zhaolin Long, Shoujie Li, Dan Zhou, Ying Jin, Li Zhang
Correspondence: Wei Cheng(chengwei669@scu.edu.cn)

Methanobactins (Mbns) are a family of copper-binding peptides involved in copper uptake by methanotrophs, and are potential therapeutic agents for treating diseases characterized by disordered copper accumulation. Mbns are produced via modification of MbnA precursor peptides at cysteine residues catalyzed by the core biosynthetic machinery containing MbnB, an iron-dependent enzyme, and MbnC. However, mechanistic details underlying the catalysis of the MbnBC holoenzyme remain unclear. Here, we present crystal structures of MbnABC complexes from two distinct species, revealing that the leader peptide of the substrate MbnA binds MbnC for recruitment of the MbnBC holoenzyme, while the core peptide of MbnA resides in the catalytic cavity created by the MbnB–MbnC interaction which harbors a unique tri-iron cluster. Ligation of the substrate sulfhydryl group to the tri-iron center achieves a dioxygen-dependent reaction for oxazolone-thioamide installation. Structural analysis of the MbnABC complexes together with functional investigation of MbnB variants identified a conserved catalytic aspartate residue as a general base required for MbnBC-mediated MbnA modification. Together, our study reveals the similar architecture and function of MbnBC complexes from different species, demonstrating an evolutionarily conserved catalytic mechanism of the MbnBC holoenzymes.

https://doi.org/10.1038/s41422-022-00620-2

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