Identification and characterization of a bacterial core methionine synthase

Darja Deobald,Rafael Hanna,Gunhild Layer,Shahab Shahryari,Lorenz Adrian

doi:10.1038/s41598-020-58873-z

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.

Highlights

Reactivation of cob(II)alamin to methylcob(III)alamin, which is generated in a side-reaction approximately once in 2,000 turnovers[15], SAM is required[16]
Since methionine biosynthesis and SAM metabolism are biochemically closely linked, we started our search for a methionine synthase gene in the genome of strain CBDB1 by inspecting the direct neighborhood of the loci cbdbA476 and cbdbA477
Locus cbdbA481 located in the same operon encodes a 343 amino acid polypeptide with a calculated molecular mass of 38 kDa that has sequence similarity with the C-terminal half of canonical tandem-repeat methionine synthase (MetE) proteins

Summary

Introduction

Reactivation of cob(II)alamin to methylcob(III)alamin, which is generated in a side-reaction approximately once in 2,000 turnovers[15], SAM is required[16]. Canonical MetE proteins (EC 2.1.1.14) are described as a family of zinc-containing metalloenzymes sharing no sequence similarity with MetH1,13. They catalyze the methylation of homocysteine using 5-methyl-THF-Glun (n ≥ 3) as methyl donor without the involvement of cobalamin. MetE in E. coli is a protein of 753 amino acid residues (85 kDa) that is composed of two homologous parts connected by a linker region (Fig. 1b), suggesting that the domains have evolved by gene duplication of a sequence encoding a smaller protein of approximately 340. We found that locus cbdbA481 (NCBI accession number CAI82680) of D. mccartyi strain CBDB1 encodes a 343 amino acid protein that is homologous to the C-terminus of canonical tr-MetE and similar to methylcobalamin:homocysteine methyltransferase (core-MetEMTH) of the methanogenic archaeum Methanobacterium thermoautotrophicum[33]. Together with archaeal methylcobalamin:homocysteine methyltransferases and bacterial homologs the gene product of locus cbdbA481 forms a new group of basal methionine synthases, referred to as core-MetE in the following

Methods

Results

Conclusion