Abstract
By catalysing the microbial formation of methane, methyl-coenzyme M reductase has a central role in the global levels of this greenhouse gas1,2. The activity of methyl-coenzyme M reductase is profoundly affected by several unique post-translational modifications3–6, such as a unique C-methylation reaction catalysed by methanogenesis marker protein 10 (Mmp10), a radical S-adenosyl-l-methionine (SAM) enzyme7,8. Here we report the spectroscopic investigation and atomic resolution structure of Mmp10 from Methanosarcina acetivorans, a unique B12 (cobalamin)-dependent radical SAM enzyme9. The structure of Mmp10 reveals a unique enzyme architecture with four metallic centres and critical structural features involved in the control of catalysis. In addition, the structure of the enzyme–substrate complex offers a glimpse into a B12-dependent radical SAM enzyme in a precatalytic state. By combining electron paramagnetic resonance spectroscopy, structural biology and biochemistry, our study illuminates the mechanism by which the emerging superfamily of B12-dependent radical SAM enzymes catalyse chemically challenging alkylation reactions and identifies distinctive active site rearrangements to provide a structural rationale for the dual use of the SAM cofactor for radical and nucleophilic chemistry.
Highlights
7-thioheptanoylthreoninephosphate (CoB) to a CoB–CoM heterodisulfide and methane (Fig. 1a)
Mmp[10], which has been shown to catalyse this key post-translational modification[7,8], belongs to an emerging superfamily of B12-dependent radical SAM enzymes[13,14,15,16,17,18,19,20,21] that encompasses more than 200,000 proteins[22]. These enzymes are involved in the biosynthesis of myriad natural products including bacteriochlorophyll and antibiotics[9,16,18,23] and catalyse various reactions such as methyl transfer to sp2- and sp3-hybridized carbon atoms[13,14,18,24], P-methylation[25], ring contraction and cyclization reactions[26,27]
One molecule each of 5′-deoxyadenosine (5′-dA) and SAH were produced per methylation reaction (Extended Data Fig. 3b), whereas no SAM cleavage was noted in the absence of peptide, regardless of the reductant used
Summary
No notable overall structural change was observed when Mmp[10] was co-crystallized with the demethylated SAM product, with a root mean squared deviation (r.m.s.d.) of 0.37 Å over 408 residues; SAM adenine binding remained mostly unaffected. Mmp[10] adopted a closed conformation involving displacement of the α1a-helix by 11.6 Å and the α1- to α4-helices of the radical SAM TIM barrel by as much as 3.4 Å (Extended Data Fig. 5a, b). Entrance of the active site, the peptide backbone formed a sharp twist assisted by two conserved proline residues and a complex network of polar interactions between charged amino acid side chains and the enzyme backbone (D6, Y56, E54 and G87) (Extended Data Fig. 5c). Its presence appears to be essential for preventing major backbone reorganization during Y115 motion, as mutation of D156, which makes key interactions in the cation-binding site, severely impairs enzyme activity (Extended Data Fig. 7b, c). These bonds are critical for the interface between the radical SAM and cobalamin-binding domains and are necessary for strict control of catalysis
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