L-methionine γ-lyase from Citrobacter freundii: Cloning of the gene and kinetic parameters of the enzyme

Abstract

It is shown for the first time for the Enterobacteriaceae family that a gene encoding L-methionine gamma-lyase (MGL) is present in the genome of Citrobacter freundii. Homogeneous enzyme has been purified from C. freundii cells and its N-terminal sequence has been determined. The hybrid plasmid pUCmgl obtained from the C. freundii genomic library contains an EcoRI insert of about 3000 bp, which ensures the appearance of MGL activity when expressed in Escherichia coli TG1 cells. The nucleotide sequence of the EcoRI fragment contains two open reading frames. The first frame (the megL gene) encodes a protein of 398 amino acid residues that has sequence homology with MGLs from different sources. The second frame encodes a protein with sequence homology with proteins belonging to the family of permeases. To overexpress the megL gene it was cloned into pET-15b vector. Recombinant enzyme has been purified and its kinetic parameters have been determined. It is demonstrated that a presence of a hybrid plasmid pUCmgl, containing the megL gene in the E. coli K12 cells, leads to a decrease in efficiency of EcoKI-restriction. It seems likely that decomposition of L-methionine under the action of MGL leads to a decrease in the intracellular content of S-adenosylmethionine. Expression of the megL gene in the C. freundii genome occurs only upon induction by a significant amount of L-methionine.

Global Regulation of the Response to Sulfur Availability in the Cheese-Related Bacterium Brevibacterium aurantiacum

“…The methionine ␥-lyase is the key enzyme for methionine degradation in Brevibacteriaceae and other bacteria (25,32). The expression of genes encoding methionine ␥-lyases is induced by methionine in Pseudomonas putida (25) and Citrobacter freundii (31). Methionine ␥-lyase activity in B. antiquum CNRZ918 (formerly B. linens) also increases in the presence of methionine (18).…”

“…BL1655 to BL1657 and BL1293 to BL1296 share similarities with MetNPQ, a high-affinity ABC transporter of B. subtilis (53,35, and 36% identities) (48) and a cystine ABC transporter of E. coli (48,31,33, and 26% identities), respectively. Other putative cystine or methionine transporters are present in the B. aurantiacum genome.…”

Biocatalytic Cascade Reaction for the Asymmetric Synthesis of L‐ and D‐Homoalanine

“…PLP concentration did not show a significant influence on the final conversion (see Supporting Information for details). These results are corroborated by previous studies in literature where METases with optimal activities at 37 °C and pH 7 were described.…”

High-resolution structure of methionine γ-lyase fromCitrobacter freundii

“…In the eukaryotic pathogens Entamoeba histolytica (Tokoro et al, 2003) and Trichomonas vaginalis (McKie et al, 1998), two genes encode MGL enzymes with a sequence identity of around 70% and with differing kinetic properties. Data on the substrate-and reaction-specificity of the enzyme are mainly restricted to the MGLs from Pseudomonas putida (Esaki et al, 1977(Esaki et al, , 1979Tanaka et al, 1985;Inoue et al, 2000;Takakura et al, 2004), T. vaginalis (Lockwood & Coombs, 1991;McKie et al, 1998), E. histolytica (Tokoro et al, 2003;Sato et al, 2006) and C. freundii (Manukhov et al, 2006;Alferov et al, 2006). The mechanisms of theand -elimination reactions catalyzed by MGL are not well understood compared with those of other PLP-dependent enzymes, particularly aminotransferases (Eliot & Kirsch, 2004).…”