YtjE from Lactococcus lactis IL1403 Is a C-S lyase with alpha, gamma-elimination activity toward methionine

Abstract

Cheese microbiota and the enzymatic conversion of methionine to volatile sulfur compounds (VSCs) are important factors in flavor formation during cheese ripening and the foci in biotechnological approaches to flavor improvement. The product of ytjE of Lactococcus lactis IL1403, suggested to be a methionine-specific aminotransferase based on genome sequence analysis, was therefore investigated for its role in methionine catabolism. The ytjE gene from Lactococcus lactis IL1403 was cloned in Escherichia coli and overexpressed and purified as a recombinant protein. When tested, the YtjE protein did not exhibit a specific methionine aminotransferase activity. Instead, YtjE exhibited C-S lyase activity and shared homology with the MalY/PatC family of enzymes involved in the degradation of L-cysteine, L-cystine, and L-cystathionine. YtjE was also shown to exhibit alpha,gamma-elimination activity toward L-methionine. In addition, gas chromatographic-mass spectrometry analysis showed that YtjE activity resulted in the formation of H2S from L-cysteine and methanethiol (and its oxidized derivatives dimethyl disulfide and dimethyl trisulfide) from L-methionine. Given their significance in cheese flavor development, VSC production by YtjE could offer an additional approach for the development of cultures with optimized aromatic properties.

FIG. 1.

SDS-PAGE analysis of CFEs prepared from an IPTG-induced culture of E. coli pET28-ytjE (lanes: 4, total fraction; 5, soluble fraction; 6, insoluble fraction). CFEs prepared from an IPTG-induced culture of E. coli carrying empty vector (lanes: 1, total fraction; 2, soluble fraction; 3, insoluble fraction) were included as a control. Purified His-tagged YtjE protein is also shown (arrow; lane 7). The molecular mass markers are also indicated (kDa).

FIG. 2.

Alignment of YtjE amino acid sequence from L. lactis IL1403 with Lcd from S. anginosus, PatB from B. subtilis, PatC from L. delbrueckii, and MalY from E. coli. Boxes indicate identical amino acid residues in all the sequences analyzed. The four residues that are invariant in all aminotransferases (▾) (1) are shown. The Lys₂₃₃ residue is the potential binding site of PLP. Residues typical for aminotransferases (•) and those typical for trans-sulfuration enzymes (○) are also indicated.

FIG. 3.

Cysteine desulfhydrase activity by in situ staining. YtjE activity was monitored in Tris-glycine gels under nondenaturing conditions, using l-cysteine as the substrate. Lanes: 1, 5 μg purified recombinant protein; 2, 2.5 μg purified recombinant protein; 3, CFE from E. coli recombinant strain expressing YtjE; 4, CFE from E. coli carrying empty vector; 5, CFE from L. lactis IL1403.

FIG. 4.

Production of MTL, DMDS, and DMTS by coincubating l-methionine with the purified YtjE protein. Values are the means of three independent measurements. The standard errors of the means are also indicated.