Structures in Tetrahydrofolate Methylation in Desulfitobacterial Glycine Betaine Metabolism at Atomic Resolution

Abstract Enzymes orchestrating methylation between tetrahydrofolate (THF) and cobalamin (Cbl) are abundant among all domains of life. During energy production in Desulfitobacterium hafniense, MtgA catalyzes the methyl transfer from methylcobalamin (Cbl‐CH3) to THF in the catabolism of glycine betaine (GB). Despite its lack of sequence identity with known structures, we could show that MtgA forms a homodimeric complex of two TIM barrels. Atomic crystallographic insights into the interplay of MtgA with THF as well as analysis of a trapped reaction intermediate (THF‐CH3)+ reveal conformational rearrangements during the transfer reaction. Whereas residues for THF methylation are conserved, the binding mode for the THF glutamyl‐p‐aminobenzoate moiety (THF tail) is unique. Apart from snapshots of individual reaction steps of MtgA, structure‐based mutagenesis combined with enzymatic activity assays allowed a mechanistic description of the methyl transfer between Cbl‐CH3 and THF. Altogether, the THF‐tail‐binding motion observed in MtgA is unique compared to other THF methyltransferases and therefore contributes to the general understanding of THF‐mediated methyl transfer.

Enzymes orchestrating methylationb etween tetrahydrofolate (THF) and cobalamin (Cbl) are abundant amonga ll domainso f life. During energy production in Desulfitobacterium hafniense, MtgA catalyzes the methyl transfer from methylcobalamin (Cbl-CH 3 )t oT HF in the catabolism of glycineb etaine (GB). Despite its lack of sequence identityw ith knowns tructures, we could show that MtgA forms ah omodimeric complex of two TIM barrels. Atomic crystallographic insights into the interplay of MtgA with THF as well as analysis of at rapped reaction intermediate (THF-CH 3 ) + reveal conformationalr earrangements during the transfer reaction. Whereas residues for THF methylation are conserved, the binding mode for the THF glutamyl-paminobenzoatem oiety (THF tail) is unique.A part from snapshots of individual reaction steps of MtgA, structure-based mutagenesis combined with enzymatic activity assays allowed am echanisticd escription of the methyl transfer between Cbl-CH 3 and THF.A ltogether,t he THF-tail-binding motion observed in MtgA is unique compared to other THF methyltransferases and therefore contributes to the general understanding of THF-mediated methyl transfer.
Enzymatic reactions involvingt he cofactor tetrahydrofolate (THF) are found ubiquitouslyt hroughout nature and support varioust ransfer reactions of reduced single carbon species. [1] THF methyl transfer is often dependento nc obalamin (Cbl), combining as implistic reaction with two highly sophisticated cofactors. [2] This raises the questions of how the transfer of the methyl group is carried out mechanistically and why such cofactor complexity is required.
The most prominent example of methyl transfer from THF to Cbl takes place in methionineb iosynthesis, where the CH 3 group from N 5 -methyltetrahydrofolate (THF-CH 3 )i su sed to convert homocysteine into methionine via methylcob(III)alamin (Cbl-CH 3 ). [3] Yet, THF-and Cbl-coupled methyl transfer has been shown to be widely distributed. In the Wood-Ljungdahl pathway,a cetogenic anaerobic microbes can grow on CO 2 and methylated substrates by producing acetyl-CoA by THF-and Cbl-coupled methyl transfer. [4] In addition, variations of these Cbl-dependentenzymesystems exist that accommodate different methyl-groups ourcesa nd acceptor moleculesf or specific metabolic challenges. [5] For instance, the O-demethylation of phenyl methyl ethers as well as N-demethylation of the quaternary amine glycine betaine (GB) are used for energy production as well as carbon assimilation in acetogenicb acteria. [6] Desulfitobacteriumh afniense,anon-acetogenic organism,h as been found to employ these precursors solelyf or energy productiont hrough anaerobic respiration. [7] In the case of the GB pathway,t his organism uses as eries of enzymes,M tgB, MtgC, and MtgA, to transfero ne methylg roup via Cblt oT HF (Scheme 1). [8] MtgA catalyzesm ethyl transfer from Cbl-CH 3 to THF and lacks sequence identityt owards solved protein structures. [9][10][11] Herein, we present X-ray structures of THF-and THF-CH 3 -bound MtgA that allow us atomici nsights into the reaction trajectory of this catalyst. Site-directed mutagenesis combined with activity assays identifiedauniquel ocking mode of the THF glutamyl-p-aminobenzoatemoiety (THF tail).
MtgA was cloned and heterologously expressed in Escherichia coli K12. The enzyme was purified to homogeneity and co-crystallized with THF and THF-CH 3 ( Figure S1, Ta bles S1 and S2 in the SupportingI nformation). Structure elucidation was performed by experimentalp hasing with selenomethionine-labeled protein. THF and THF-CH 3 -bound complexes were solved at ar esolutions of 1.35 (PDB ID:6 SJ8) and 1.55 (PDB ID: 6SK4), respectively (Table S3). MtgA assembles as ah omodimer with ac ontact area of 1870 2 ; [12] this agreesw ith retention times from size-exclusion chromatography.T he topology of each subunit is characterized by af unnel of eightp arallel bsheetsc onnected by a-helices on the outside that form ac omplex of two perpendicularly oriented TIM barrels ( Figure 1A). [13] The structure contains two additional small antiparallel bsheetsa tt he Nterminus. Compared to the typical (b/a) 8 architecture, b-sheet 7i nM tgA is replaced by ar andom coil and two small a-helices ranging from residue 218 to 238 (Figure S2). The active-site cavity is located inside the hydrophilic core of each barrel ( Figure 1B).
Intriguingly,t he X-ray structureo ft he MtgA:THF complex illustrates electron density only for the pterin moiety of the ligand;the THF tail seems to be flexible ( Figure 1C). The identified binding site of MtgA consists of ther esidues Ser32, Asp75, Asp102,A sn129 andA sp194,w hich stabilizet he aromatic ring system of THF by hydrogen bonds. The close proximity of the acidic residues Asp75 and Asp102 towards each other is most striking. Hereby,t he strong interaction of Asp102w ith the co- Scheme1.To pl eft:Physiological pathways involving methylt ransfer between Cbl and THF.Y ellowb ox:Inmicrobial glycine betaine (GB) metabolism, the methylgroup( blue) is abstractedf rom GB and transferred to the N 5 atom of THF.InD. hafniense,THF-CH 3 is formedb yM tgA (pink) and furthercatabolized for energy production. [6,8] The glutamyl-p-aminobenzoate moiety of THF is abbreviated as R. factor increases the negative partial charge at N 5 of THF and activatesi tf or attack of the incoming Cbl-bound CH 3 group. These first mechanistic insights were confirmed by replacing Asp102 with alanine. In the MtgA D102A mutant, methyl transfer from Cbl-CH 3 to THF is abolished as compared to the wildtype (WT) enzyme (Figure2B).
In contrast to substrate-bound MtgA, the entire cofactor is defined in the MtgA:THF-CH 3 complex depicting extensive coordination within the amino acid environment in ar igid manner( Figure 1D). The overall bindingm otif of the pterin ring remains similart ot he substrate-bound structure (Figure 1C). However,t he THF benzoate moiety is now involved in a p-stacking network including Tyr230, Phe243, Phe251, and Trp272 on one side, as well as Tyr35 and His38 on the other. While the functional groups of the THF tail engagew ater-coupled hydrogen bonding interactions with adjacent residues, the terminal g-carboxylate forms salt bridges with Arg236 that coordinate the cofactor in an extended conformation ( Figure 1D). Mutation of Arg236t oa laniner esulted in impaired enzymatic activity ( Figure 2B), thus implying that fixation of the THF tail during methylation is involved in catalysis. Besides this fixation, the superposition of both ligand structures illustrates ar otation of the pterin moiety during the reaction of approximately 358.I nb oth poses, the aromaticr ing system is well stabilized andm ost active-site protein residues remaini n their original position ( Figure 1E). In contrast, the benzoate group of THF-CH 3 displaces the carboxamide side chain of Asn227b y2 ( Figure 1E,r ed double arrow).T his conformation shift causes helix a7' to collapse ( Figure S2), which is compensated for by the formationo fa dditional hydrogen bonds between N 5 ,A sn227, and Ser226 ( Figure 1E,y ellow dotted line). Thus,t he replacement of the typical TIM barrel b-sheet 7 with helix a7' (Figure S2) appearstobeanecessary adjustment for efficient catalysis in MtgA.
In the MtgA:THF-CH 3 complex, the cofactor exhibits tetrahedral sp 3 hybridizationa ti ts methylated amine N 5 .A sp lanar sp 2 hybridization would be expected in this positiono ft he final product THF-CH 3 ,t he X-ray structurem ust depictat rapped protonated reaction intermediate ( Figure 1D,E ). As ar esult of its performed swinging motion, Asn227i si np osition to form a strong hydrogen bond with the N 5 atom of (THF-CH 3 ) + .N otably,t he active site lacks residues to deprotonatet he (THF-CH 3 ) + intermediate;t his decreases the conjugatede lectron system within the pterin ring system. Supported by the inactive N227A and N227D mutants, we propose that proper stabilization of the tetrahedral N 5 state of (THF-CH 3 ) + is fundamental forcatalyzing the methyl transfer. (Figure 2B).
X-ray structure analysis of mutant MtgA in complex with THF-CH 3 gave further insights into the reaction trajectory. Whereas the N227A:THF-CH 3 complex (1.8 ,P DB ID:6 SJS) features aw ater molecule in place of the carboxamide side chain as well as planar sp 2 hybridization at N 5 (Figure S4 B), the D102A:THF-CH 3 structure (1.95 ,P DB ID:6 SJO) displays undefinede lectron density around N 5 ,t hus indicating fluctuation between sp 2 and activateds p 3 hybridization (Figure S4 A). Therefore, both mutants mimic product formation at al ate reaction state. On the other hand, the WT MtgA:THF-CH 3 structure illustrates an intermediate that formsi mmediatelya fter CH 3 transfer from Cbl-CH 3 to THF (Figures 1D and S4). Altogether, the presentedc rystallographic snapshots depict enzyme catalysis in action at atomicr esolution.
Despite its individual sequence, MtgA shares the TIM barrel fold with ac ouple of Cbl-dependentT HF methyltransferases. These include methionine synthase (MetH) and the THF-binding component from the carbon-fixating Wood-Ljungdahl pathway in acetogenic bacteria (MeTr). [10,11,14,15] The desulfitobacterialm ethyl group acceptorp rotein MT2DH, which plays a role in the catabolism of aromatic methyl ethers (Scheme1), showst he highest structurals imilarity with MtgA (r.m.s.d.: 2.9 ,s equence identity:1 3%, Z score:2 0.2;F igure S3). [9,16] In agreement with MT2DH andc ontrast to MetH and MeTr, MtgA catalyzes methyl transfer from Cbl-CH 3 to THF (Scheme 1A). Regardless of the direction of methyl transfer,r esidues involvedi nc atalysis and binding of the pterin ring are conserved. Moreover,ap rotonated THF intermediate seems to be acommon feature of Cbl-dependent THF methyltransferases. [17] Nonetheless, MtgA has unique features. Whereas MT2DH lacks conformational rearrangements between THF and THF-CH 3bound states, [9] the catalytic Asn of the more distantly related MeTra cts as gatekeeper for Cbl prior to entry of THF-CH 3 . [15] In MtgA, this Asn is involved in as winging motion that is key to recognizing the proper folate cofactor and catalyzing the  transfer to its N 5 atom. Even though thesee nzymes share mechanistic principles, structural peculiarities are essential for efficient catalysise ither in the catabolism of GB or other Cbl-dependentT HF methyl-transfer pathways. Ta ken together,s tructural and biological characterizationo fM tgA supports the general understanding of biological systemse mploying the cofactor THF as atool for methylation reactions.