Identification and characterization of a bacterial core methionine synthase

Abstract

Methionine synthases are essential enzymes for amino acid and methyl group metabolism in all domains of life. Here, we describe a putatively anciently derived type of methionine synthase yet unknown in bacteria, here referred to as core-MetE. The enzyme appears to represent a minimal MetE form and transfers methyl groups from methylcobalamin instead of methyl-tetrahydrofolate to homocysteine. Accordingly, it does not possess the tetrahydrofolate binding domain described for canonical bacterial MetE proteins. In Dehalococcoides mccartyi strain CBDB1, an obligate anaerobic, mesophilic, slowly growing organohalide-respiring bacterium, it is encoded by the locus cbdbA481. In line with the observation to not accept methyl groups from methyl-tetrahydrofolate, all known genomes of bacteria of the class Dehalococcoidia lack metF encoding for methylene-tetrahydrofolate reductase synthesizing methyl-tetrahydrofolate, but all contain a core-metE gene. We heterologously expressed core-MetE CBDB in E. coli and purified the 38 kDa protein. Core-MetE CBDB exhibited Michaelis-Menten kinetics with respect to methylcob(III)alamin (K M ≈ 240 µM) and L-homocysteine (K M ≈ 50 µM). Only methylcob(III)alamin was found to be active as methyl donor with a k cat ≈ 60 s −1. Core-MetE CBDB did not functionally complement metE-deficient E. coli strain DH5α (ΔmetE::kan) suggesting that core-MetE CBDB and the canonical MetE enzyme from E. coli have different enzymatic specificities also in vivo. Core-MetE appears to be similar to a MetE-ancestor evolved before LUCA (last universal common ancestor) using methylated cobalamins as methyl donor whereas the canonical MetE consists of a tandem repeat and might have evolved by duplication of the core-MetE and diversification of the N-terminal part to a tetrahydrofolate-binding domain. Methionine plays an essential role as proteinogenic amino acid in all domains of life, as an initiation amino acid in protein translation 1 and as a precursor in the formation of cysteine, carnitine, taurine and lecithin 2,3. Moreover, methionine can be converted to S-adenosyl-L-methionine (SAM) 4 , which represents an activated methyl group donor for many fundamental cellular processes 5,6. The final step in methionine de novo synthesis, the methylation of homocysteine to methionine, is catalyzed by different types of methionine synthases including cobalamin-dependent (MetH) and cobalamin-independent methionine synthase (MetE). Some bacteria, e.g. Escherichia coli, possess genes for both enzymes 7 and repress the expression of metE in the presence of vitamin B 12 8. Homocysteine methylation in mammals is catalyzed by mammalian methionine synthases (mMS) similar to bacterial MetH 9 , betaine-L-homocysteine-S-methyltransferase (BHMT) or S-methyl-L-methionine-L-homocysteine-S-methyltransferase (also known as BHMT-2) 10. Fungi and plants encode exclusively MetE 11 or BHMT-2 12. All known methionine synthase types contain a zinc ion in the active site that is essential for homocysteine binding and methyl group transfer 13. ...

One‐carbon metabolism, folate, zinc and translation

“…A general study, focused on plants but with some comparisons with S. cerevisiae and E. coli and animals, showed a variety of trees, which, again, would lend credence to widespread HGT, precluding faithful identification of relevant rooting (Lian et al , 2015). In this context, the recent identification of a methyl‐B12‐dependent, H 4 F‐independent form of the enzyme should be interpreted as a recent acquisition (synthesis of coenzyme B12, requiring at least 30 genes, is hardly an early feature of life), rather than primitive (Deobald et al , 2020).…”

Four Families of Folate-independent Methionine Synthases

“…To identify likely functional orthologs of MesB, we began with the hypothesis (Deobald et al 2020) that MesB obtains methyl groups from the CoFeSP protein of the Wood-Ljungdahl pathway ( Figure 2). This hypothesis is consistent with labeling experiments with Dehalococcoides mccartyi , which show that the methyl group of methionine is derived from the methyl group of acetate (Zhuang et al 2014) .…”

“…Besides MetH and MetE, three other types of methionine synthases have been reported. These "core" methionine synthases (Deobald et al 2020) are homologous to the C-terminal catalytic domain of MetE and do not contain any other domains. In particular, they lack the N-terminal domain of MetE that is involved in binding folate (Pejchal and Ludwig 2005) .…”

“…Second, a core methionine synthase from the bacterium Dehalococcoides mccartyi was recently identified (Deobald et al 2020) . We will call this protein MesB (UniProt:A0A0V8M4G6).…”

“…We will call this protein MesB (UniProt:A0A0V8M4G6). MesB uses methylcobalamin, but not 5-methyl-THF, as a substrate in vitro , and MesB cannot complement a metE-strain of E. coli (Deobald et al 2020) . MesB was proposed to obtain methyl groups from the iron-sulfur corrinoid protein (CoFeSP) of the Wood-Ljungdahl pathway (Deobald et al 2020) .…”