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Cobalamin-dependent methionine synthase (MS) is a key enzyme in methionine and folate one-carbon metabolism. MS is a large multi-domain protein capable of binding and activating three substrates: homocysteine, folate, and S -adenosylmethionine for methylation. Achieving three chemically distinct methylations necessitates significant domain rearrangements to facilitate substrate access to the cobalamin cofactor at the right time. The distinct conformations required for each reaction have eluded structural characterization as its inherently dynamic nature renders structural studies difficult. Here, we use a thermophilic MS homolog ( t MS) as a functional MS model. Its exceptional stability enabled characterization of MS in the absence of cobalamin, marking the only studies of a cobalamin-binding protein in its apoenzyme state. More importantly, we report the high-resolution full-length MS structure, ending a multi-decade quest. We also capture cobalamin loading in crystallo , providing structural insights into holoenzyme formation. Our work paves the way for unraveling how MS orchestrates large-scale domain rearrangements crucial for achieving challenging chemistries. Methionine synthase (MS) harnesses B12 and flexibility to catalyze three different reactions on one protein. The full-length structure of MS yields insights into a protein that epitomizes controlled dynamics to dictate chemical outcome.

Itaconate is an immunometabolite with both anti-inflammatory and bactericidal effects. Its coenzyme A (CoA) derivative, itaconyl-CoA, inhibits B12-dependent methylmalonyl-CoA mutase (MCM) by an unknown mechanism. We demonstrate that itaconyl-CoA is a suicide inactivator of human and Mycobacterium tuberculosis MCM, which forms a markedly air-stable biradical adduct with the 5′-deoxyadenosyl moiety of the B12 coenzyme. Termination of the catalytic cycle in this way impairs communication between MCM and its auxiliary repair proteins. Crystallography and spectroscopy of the inhibited enzyme are consistent with a metal-centered cobalt radical ~6 angstroms away from the tertiary carbon-centered radical and suggest a means of controlling radical trajectories during MCM catalysis. Mycobacterial MCM thus joins enzymes in the glyoxylate shunt and the methylcitrate cycle as targets of itaconate in pathogen propionate metabolism.

Pseudouridine (Ψ) is a ubiquitous RNA modification incorporated by pseudouridine synthase (Pus) enzymes into hundreds of noncoding and protein-coding RNA substrates. Here, we determined the contributions of substrate structure and protein sequence to binding and catalysis by pseudouridine synthase 7 (Pus7), one of the principal messenger RNA (mRNA) modifying enzymes. Pus7 is distinct among the eukaryotic Pus proteins because it modifies a wider variety of substrates and shares limited homology with other Pus family members. We solved the crystal structure of Saccharomyces cerevisiae Pus7, detailing the architecture of the eukaryotic-specific insertions thought to be responsible for the expanded substrate scope of Pus7. Additionally, we identified an insertion domain in the protein that fine-tunes Pus7 activity both in vitro and in cells. These data demonstrate that Pus7 preferentially binds substrates possessing the previously identified UGU̱AR (R = purine) consensus sequence and that RNA secondary structure is not a strong requirement for Pus7-binding. In contrast, the rate constants and extent of Ψ incorporation are more influenced by RNA structure, with Pus7 modifying UGU̱AR sequences in less-structured contexts more efficiently both in vitro and in cells. Although less-structured substrates were preferred, Pus7 fully modified every transfer RNA, mRNA, and nonnatural RNA containing the consensus recognition sequence that we tested. Our findings suggest that Pus7 is a promiscuous enzyme and lead us to propose that factors beyond inherent enzyme properties (e.g., enzyme localization, RNA structure, and competition with other RNA-binding proteins) largely dictate Pus7 substrate selection.

Enzymes are Nature’s best chemists’ and play a vital role in supporting the diverse chemistries fundamental to complex life. Given this central role, protein dysfunction can have serious biological implications. Proteins are defined by the Structure-Function relationship ubiquitous throughout nature, and these relationships can be exploited for detailed enzymatic characterization. Thus, I took an integrative structural and biochemical approach to establish the structural context and molecular basis of challenging chemistries catalyzed by three enzymes involved in mRNA modification and cobalamin-dependent processes, and whose dysfunction result in cancers, developmental- and metabolic disorders.Pseudouridine (Ψ) is a ubiquitous RNA modification, discovered at hundreds of sites in mRNAs. Pseudouridine synthases (Pus) are responsible for installing Ψ, but exactly how an individual Pus selects a specific target site is unclear. I sought to characterize the basis of Pus-RNA interactions in Pus7 and ultimately determined the contribution of substrate structure and conserved protein elements towards binding and catalysis. Pus7 is one of the predominant mRNA modifying Pus-enzymes, that exhibits distinct diversity in substrate selectivity, as well as increased activity under heat shock. I solved the structure of Saccharomyces cerevisiae Pus7 and visualized the architecture of the eukaryotic-specific insertions thought to contribute to expanded substrate scope. Indeed, the largest insertion (Insertion I) contains a nucleic acid binding R3H motif surrounded by positively charged residues. Subsequent analysis demonstrated that Insertion-I serves to fine-tune Pus7 activity in a substrate-dependent manner both in vitro and in cells. Further, this work revealed that Pus7 is extraordinarily promiscuous, modifying every substrate (both natural and non-natural) containing the consensus sequence without regard for structure. My work suggests that Pus7 selectivity is likely governed by additional factors including substrate accessibility and localization, rather than inherent enzyme properties.B12-dependent enzymes harness the unique organometallic properties of cobalt to catalyze a variety of challenging chemistries integral to single-carbon metabolism in all domains of life. In humans, there are two metabolically essential B12-dependent enzymes: methionine synthase (MS) and methylmalonyl-CoA mutase (MCM). Cobalamin-dependent MS is a multi-modular enzyme that employs remarkable molecular dynamics and domain rearrangements – deemed ‘molecular juggling’ – to catalyze three difficult methyl-transfer reactions at the site of the cobalt-cofactor. Biochemical challenges have hindered structural and mechanistic characterization of MS catalysis and conformational states. To address this, I describe a Thermus thermophilus MS variant that avoids the associated barriers of expression and purification. Using tMS as a model, I describe the first full-length structure of apoMS – finally visualizing all domains at once and gaining insights into the structural basis of B12-incorporation. Further, we captured MS with the Folate-domain oriented above the B12-domain, and cobalt is within the predicted distance for catalysis, and this likely represents the first catalytic structure captured for any corrinoid protein.MCM utilizes 5’-deoxyadenosylcobalamin (AdoCbl) to catalyze the interconversion of methylmalonyl-CoA to succinyl-CoA through homolysis of Co-C bond. Here, we determined the structure of Mycobacterium tuberculous MCM complexed with the suicide inactivator itaconyl-CoA, a succinyl-CoA analog. Notably, EPR studies confirm that we captured an air-stable biradical comprising a tertiary carbon radical (5’-deoxyadenosyl) coupled to the metal-centered cob(II)alamin radical in crystallo. Thus, in addition to describing the mechanism of I-CoA inhibition, these experiments provide molecular insights into how MCM controls radical trajectories during catalysis.

Cobalamin-dependent methionine synthase (MS) is a key enzyme in methionine and folate one-carbon metabolism. MS is a large multi-domain protein capable of binding and activating three substrates: homocysteine, folate, S-adenosylmethionine for methylation. Achieving three chemically distinct methylations necessitates significant domain rearrangements to facilitate substrate access to the cobalamin cofactor at the right time. The distinct conformations required for each reaction have eluded structural characterization as its inherently dynamic nature renders structural studies difficult. Here, we use a thermophilic MS homolog (tMS) as a functional MS model. Its exceptional stability enabled characterization of MS in the absence of cobalamin, marking the first studies of a cobalamin-binding protein in its apoenzyme state. More importantly, we report the first high-resolution full-length MS structure, ending a multi-decade quest. We also captured cobalamin loading in crystallo, providing structural insights into holoenzyme formation. Our work paves the way for unraveling how MS orchestrates large-scale domain rearrangements crucial for achieving challenging chemistries.