Chinese Study Discovers Crystal Structure And Catalytic Mechanism Of MbnBC Holoenzyme Required For Methanotrophic Biosynthesis

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Methanobactins (Mbns) are a family of copper-binding peptides involved in the uptake of copper by methanotrophs and are potential therapeutic agents for the treatment of diseases characterized by disordered copper accumulation. Mbns are generated by modification of the MbnA precursor peptide on cysteine residues catalyzed by the core biosynthetic machinery comprising MbnB, iron-dependent enzymes, and MbnC. However, the mechanistic details of MbnBC holoenzyme catalysis remain unclear.

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On February 2, 2022, a research paper titled "Crystal structure and catalytic mechanism of the MbnBC holoenzyme required for methanobactin biosynthesis" was published online in Cell Research by Cheng Wei's team from Sichuan University, which demonstrated MbnABC complexes from two different species. The crystal structure of the substrate revealed that the leader peptide of the substrate MbnA binds to MbnC to recruit the MbnBC holoenzyme, while the core peptide of MbnA is located in the catalytic cavity created by the MbnB-MbnC interaction and possesses a unique triiron cluster.

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The attachment of the substrate sulfhydryl to the triiron center enables a dioxygen-dependent reaction for oxazolone-thioamide installation. Structural analysis of the MbnABC complex and functional studies of MbnB variants identified a conserved catalytic aspartate residue as required for MbnBC-mediated modification of MbnA. In conclusion, this study reveals similar structures and functions of MbnBC complexes from different species, demonstrating an evolutionarily conserved catalytic mechanism of the MbnBC holoenzyme.

Metals such as copper are essential for maintaining physiological homeostasis in all organisms and are particularly involved in the catalysis of some essential bacterial proteins. Although copper is required for the activity of certain proteins, it is also used as an antimicrobial agent in a variety of industrial and medical fields. In response, bacteria can be detoxified using copper-chelating compounds such as Chalkophore. Chalkophore molecules resemble iron-binding carriers involved in bacterial metabolism and detoxification. Notably, they have been studied in clinical trials as potential therapeutics for Wilson's disease, a genetic disorder that causes excessive accumulation of copper in organs (liver and brain).

Methanoxins (Mbns) are Chalkophore exclusively present in methanotrophs. Several Mbns have been characterized and found to be ribosomally synthesized and post-translationally modified peptides (RiPPs). Mbn molecules are biosynthesized from genetically encoded precursor polypeptides (MbnAs), which contain a leader peptide (LP) for recognition by the biosynthetic machinery and a core peptide (CP) that becomes the mature product after MbnA modification and LP cleavage. A core biosynthetic machinery involving the MbnBC complex was recently discovered in Methylosinus trichosporium (Mt) OB3b.

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To date, five gene-encoding clusters (I–V) have been characterized for Mbn biosynthesis. In known Mbn operons, mbnA is always followed by the mbnB and mbnC genes responsible for MbnA modification. mbnB is predicted to encode a triosephosphate isomerase (TIM) and displays MbnA-modifying activity only when complexed with MbnC. However, the organization and catalytic mechanisms of the MbnBC machinery remain unknown.

Here, the study determined the crystal structures of the RrMbnABC and VcMbnABC complexes of Rugamonas rubra (Rr) ATCC 43154 (RrMbnBC, representing group III) and Vibrio caribbenthicus (Vc) BAA-2122 (VcMbnBC, representing group V). The structures of RrMbnABC and VcMbnABC are conserved and contain a unique triiron cluster linked to cysteine residues of the substrate MbnA. The results of this study elucidate the mechanism by which the MbnBC holoenzyme catalyzes the production of Mbn.

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