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, 10 (1), 2100

Identification and Characterization of a Bacterial Core Methionine Synthase

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Identification and Characterization of a Bacterial Core Methionine Synthase

Darja Deobald et al. Sci Rep.

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-MetECBDB in E. coli and purified the 38 kDa protein. Core-MetECBDB exhibited Michaelis-Menten kinetics with respect to methylcob(III)alamin (KM ≈ 240 µM) and L-homocysteine (KM ≈ 50 µM). Only methylcob(III)alamin was found to be active as methyl donor with a kcat ≈ 60 s-1. Core-MetECBDB did not functionally complement metE-deficient E. coli strain DH5α (ΔmetE::kan) suggesting that core-MetECBDB 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.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Bioinformatic analysis of the core-MetECBDB from Dehalococcoides mccartyi strain CBDB1. (a) Maximum-Likelihood phylogenetic tree of MetE representatives was generated with MEGA7. Multiple amino acid sequence alignments of full length with deletion of gaps (MUSCLE algorithm) were used to generate the tree. The analysis involved 39 amino acid sequences including the C-terminus of tandem-repeat methionine synthases (tr-MetE) from bacteria (brown colors) and yeast (green) as well as core-MetEs from archaea (blue colors), Chloroflexi (red) and Clostridiales (brown). The gene loci are in brackets. (b) The crystal structure of core-MetECBDB was calculated with the I-TASSER server. Overlay of the crystal structure of tr-MetE from Neurospora crassa (PDB No. 4ZTX, grey) with the structural model obtained for core-MetECBDB from D. mccartyi strain CBDB1 (green) was obtained with PyMOL. Core-MetECBDB matches the C-terminal part of tr-MetENcra (C-score = −0.27) but lacks the N-terminal part and the linker region. (c) Amino acid sequence alignment of selected MetE proteins. The Zn2+-binding site HXCXnC (red) is conserved in annotated tr-MetEs and also in core-MetE homologs. Core-MetECBDB: core-MetE from D. mccartyi strain CBDB1, coreMetEDehly: core-MetE from Dehalogenimonas lykanthroporepellens, core-MetEMMKA: core-MetE from Methanococcus maripaludis, tr-MetEECDH: tandem-repeat MetE from Escherichia coli DH10B, tr-MetESau: tandem-repeat MetE from Staphylococcus aureus.
Figure 2
Figure 2
Coordination mode of methylcob(III)alamin (MeCbl(III)) bound to core-MetECBDB from Dehalococcoides mccartyi strain CBDB1. Free MeCbl(III) in the “base-on” mode is characterized by broad α/β-absorbance bands and characteristic maxima at ~ 487 and 524 nm (violet line). The UV/Vis spectrum of MeCbl(III) in the presence of core-MetECBDB at 1:1 stoichiometry was slightly changed. The absorbance intensities of β- and γ-bands increased and the maximum of the β-band shifted (Δλ = −5 nm) (red line), indicating the “base-off/His-on” binding mode of MeCbl(III).
Figure 3
Figure 3
Demethylation of methylcob(III)alamin (MeCbl(III)) catalyzed by purified core-MetECBDB from D. mccartyi strain CBDB1 in the presence of D,L-homocysteine. (a) In the presence of 0.1 µM core-MetECBDB, 0.5 mM methylcob(III)alamin and 2 mM D,L-homocysteine, continuous changes in the UV/Vis spectrum were observed indicating the consumption of methylcob(III)alamin (524 nm) and the formation of cob(I)alamin (681 nm) and cob(II)alamin (474 nm) as highlighted by arrows. (b) In the absence of core-MetECBDB, the UV/Vis spectrum remained unchanged over an incubation time of 30 min. (c) In the absence of D,L-homocysteine, the UV/Vis spectrum remained unchanged. (d) In the presence of 0.5 mg mL−1 E. coli crude extract, instead of core-MetECBDB as catalyst, MeCbl(III) was not transformed. (e) Dependence of core-MetECBDB methyltransferase activity on methylcob(III)alamin concentration. (f) Dependence of core-MetECBDB methyltransferase activity on D,L-homocysteine concentration. The activities in panels (e,f) were determined by following the change of the absorption at 681 nm. Vmax and KM were calculated according to a Hill-Fit plot with R2 = 0.998 for panel (e) and R2 = 0.999 for panel (f), respectively.
Figure 4
Figure 4
Core-MetECBDB from D. mccartyi strain CBDB1 does not catalyze the formation of L-methionine with 5-methyl-THF-Glu3 as the methyl donor. (a) Reaction described for tr-MetE from E. coli (tr-MetEEco) catalyzing the methylation of L-homocysteine with 5-methyl-THF-Glu3 to form L-methionine and THF-Glu3. (b) Representative HPLC chromatograms of a chemical standard of 5-methyl-THF-Glu3 (blue), of reaction products after an enzyme activity assay containing 5-methyl-THF-Glu3, D,L-homocysteine and either core-MetECBDB (red) or tr-MetEEco (green) or no enzyme (black). The peak at RT = 21 min represents dithiothreitol added to all reactions. In the presence of tr-MetEEco, 5-methyl-THF-Glu3 (RT = 18 min) reacts to form THF-Glu3 (RT = 17.4 min). Slow demethylation of 5-methyl-THF-Glu3 to THF-Glu3 did occur in the negative control and also in the presence of core-MetECBDB. To evaluate if this demethylation was linked to L-methionine formation, the products of the activity assays were analyzed by mass spectrometry. I) No L-methionine was formed in the presence of core-MetECBDB; II) The product of tr-MetEEco was identified as L-methionine ([M + H]+  = 150.0583 m/z).
Figure 5
Figure 5
Computational model of the active site of core-MetECBDB from Dehalococcoides mccartyi strain CBDB1. His122 is tuned towards the active site of the protein where a zinc atom is coordinated by His215, Cys217 and Cys312. His122 might replace the dimethylbenzimidazole moiety of cobalamin in the “bae-off/His-on” mode. The structure was calculated with the I-TASSER server and visualized with PyMOL.
Figure 6
Figure 6
Pathways of L-glycine and L-homocysteine methylation in Dehalococcoides species and hypothesized involvement of THF and corrinoid proteins. (a) Genes annotated to be involved in the incomplete Wood-Ljungdahl pathway encoded in D. mccartyi strain 195 and the respective homologous genes in other Dehalococcoidia. (b) L-serine formation via glycine hydroxymethyltransferase (GlyA, grey) as proposed by Zhuang et al. is shown. The methyl group is derived most probably from formate with the aid of formyl-tetrahydrofolate synthase (Fhs, yellow) and methylene-tetrahydrofolate dehydrogenase/cyclohydrolase (FolD, blue). (c) Methylation of L-homocysteine is conducted by core-MetECBDB encoded by the locus cbdbA481 (purple) in D. mccartyi strain CBDB1. The original source of the methyl group is acetate, which is activated to acetyl-CoA and then cleaved by acetyl-CoA decarbonylase (AcsB, green) into HSCoA, carbon monoxide (CO) and a methyl group. The standard activity of AcsB is to transfer the methyl group to a corrinoid iron-sulfur protein complex (CoFeSP) AcsCD (red). We speculate that the methyl group is directly transferred from the CoFeSP to the core-MetECBDB (A481) for L-homocysteine methylation (dashed arrow) but this transfer could also be indirect via a yet unidentified participant.

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References

    1. Kozak M. Initiation of translation in prokaryotes and eukaryotes. Gene. 1999;234:187–208. doi: 10.1016/S0378-1119(99)00210-3. - DOI - PubMed
    1. Stipanuk MH. Metabolism of sulfur-containing amino acids. Ann. Rev. Nutr. 1986;6:179–209. doi: 10.1146/annurev.nu.06.070186.001143. - DOI - PubMed
    1. Stipanuk MH, Ueki I. Dealing with methionine/homocysteine sulfur: cysteine metabolism to taurine and inorganic sulfur. J. Inherit. Metab. Dis. 2011;34:17–32. doi: 10.1007/s10545-009-9006-9. - DOI - PMC - PubMed
    1. Cantoni GL. S-adenosylmethionine; a new intermediate formed enzymatically from L-methionine and adenosinetriphosphate. J. Biol. Chem. 1953;204:403–416. - PubMed
    1. Gophna U, Bapteste E, Doolittle WF, Biran D, Ron EZ. Evolutionary plasticity of methionine biosynthesis. Gene. 2005;355:48–57. doi: 10.1016/j.gene.2005.05.028. - DOI - PubMed
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