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

Abstract

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.

A mixed-valent Fe(II)Fe(III) species converts cysteine to an oxazolone/thioamide pair in methanobactin biosynthesis

“…Strikingly, residues 6 to 11 of the MbnA leader peptide form an extended β sheet with four-stranded antiparallel β sheet of MbnC, interacting with MbnC residues 158 to 162 ( SI Appendix , Fig. S12 ), a finding recently confirmed by the crystal structures of Rugamonas rubra ATCC 43154 and Vibrio caribbenthicus BAA-2122 MbnBCs in complex with their cognate MbnAs ( 22 ). This interaction resembles a common RiPP precursor peptide recognition element involving the formation of an extended β sheet between a winged helix-turn-helix motif of a biosynthetic enzyme and its substrate ( 23 , 24 ).…”

Substrate Recognition by the Peptidyl-(S)-2-mercaptoglycine Synthase TglHI during 3-Thiaglutamate Biosynthesis

“…To provide a visual approximation of the TglHI complex and its interaction with the substrate, the AlphaFold-Multimer algorithm 43 was used to predict the structure of a 1:1 heterodimer of TglHI both with and without the TglACys 19mer ( Figure S4a ). In the models, TglH did not contain iron, but in the trimeric complex, the C-terminal Cys of the 19mer was still located in the vicinity of the iron ligands (based on the structure of another DUF692 family member, PDB 3BWW, and a recent study on the DUF692 enzyme MbnB 18 ). The predicted structure of apo-TglH in the complex with TglI is similar to that of MbnB, illustrating the capability of AlphaFold (the prediction was performed before the MbnB structure was reported).…”

“…MbnB contains three irons in the crystal structure, and the MbnABC complex shows threading of the substrate MbnA from MbnC to the active site of MbnB where a cysteine in the core peptide makes a direct contact with one of the irons. 18 In the predicted TglHI complex with the 19mer peptide of TglA, a very similar interaction is seen that starts at TglI and ends in the active site of TglH. In the AlphaFold model, the C-terminal domain of TglI adopts an RRE fold (three antiparallel β-strands followed by three α-helices), and the substrate TglA binds to the RRE; MbnC does not contain a canonical RRE fold, but its C-terminal domain contains a β-sheet that makes an antiparallel β-sheet interaction with the MbnA leader peptide 18 similar to the way substrate binds to β3 in RREs in other structurally characterized RiPP systems.…”

“… 18 In the predicted TglHI complex with the 19mer peptide of TglA, a very similar interaction is seen that starts at TglI and ends in the active site of TglH. In the AlphaFold model, the C-terminal domain of TglI adopts an RRE fold (three antiparallel β-strands followed by three α-helices), and the substrate TglA binds to the RRE; MbnC does not contain a canonical RRE fold, but its C-terminal domain contains a β-sheet that makes an antiparallel β-sheet interaction with the MbnA leader peptide 18 similar to the way substrate binds to β3 in RREs in other structurally characterized RiPP systems. 3 , 44 Thus, the predicted model of the 19mer binding to TglHI recaptures several of the features seen in the crystal structure of the related MbnABC protein–substrate complex.…”

“…For TglB, the minimal requirement is the final 12 amino acids, whereas TglHI requires the final 19 amino acids for catalysis, indicating overlapping but not identical substrate binding requirements. Structure prediction by AlphaFold-Multimer appears to predict the structures of TglHI well based on a recent crystal structure of MbnBC, 18 but the predicted mechanism of substrate engagement either suggests a new type of interaction between the RRE and substrate or illustrates shortcomings of the current capabilities for predicting interactions of short peptides with their modifying enzymes.…”