Cloning and characterization of two Lactobacillus casei genes encoding a cystathionine lyase

Abstract

Volatile sulfur compounds are key flavor compounds in several cheese types. To better understand the metabolism of sulfur-containing amino acids, which certainly plays a key role in the release of volatile sulfur compounds, we searched the genome database of Lactobacillus casei ATCC 334 for genes encoding putative homologs of enzymes known to degrade cysteine, cystathionine, and methionine. The search revealed that L. casei possesses two genes that putatively encode a cystathionine beta-lyase (CBL; EC 4.4.1.8). The enzyme has been implicated in the degradation of not only cystathionine but also cysteine and methionine. Recombinant CBL proteins catalyzed the degradation of L-cystathionine, O-succinyl-L-homoserine, L-cysteine, L-serine, and L-methionine to form alpha-keto acid, hydrogen sulfide, or methanethiol. The two enzymes showed notable differences in substrate specificity and pH optimum.

FIG. 1.

Cystathionine lyase activities from different L. casei genotypes measured by DTNB reaction with thiols released from l-cystathionine. Since FAM18168 was one of the strains showing the highest activity, it was always included as a reference in the assays. The mean values of three independently performed assays are shown. rel., relative; Na-P, sodium phosphate; K-P, potassium phosphate; Mes, 2-morpholinoethanesulfonic acid.

FIG. 2.

HPLC analysis of cystathionine lyase assays performed at pH 5.5 with cell extracts of L. casei ATCC 334 (left panel) and FAM18168 (right panel) genotypes. The upper panel illustrates reaction products obtained with l-cystathionine, and the lower panel shows the control (assay performed without l-cystathionine). Peaks were identified by retention time.

FIG. 3.

HPLC analysis of assays carried out at pH 5.5 with purified MalY protein (6 μg) and 2 mM l-cystathionine (A) and 2 mM l-cysteine (B) for 1 h. Peaks were identified by retention time.

FIG. 4.

HPLC analysis of assays carried out with purified MetC (9 μg) and 2 mM l-cystathionine at pH 9.0 for 1 h (A), 2 mM l-cysteine at pH 6.8 for 1 h (B), 2 mM O-succinyl-l-homoserine at pH 6.8 for 1 h (C), and 2 mM O-succinyl-l-homoserine and 2 mM l-cysteine at pH 6.8 for 1 h (D). Peaks were identified by retention time.

FIG. 5.

Headspace analysis by GC-PFPD of enzymatic assays carried out with methionine at pH 6.8. Fifty micrograms of purified protein was used for the enzymatic reaction. CS2, carbon disulfide; MTL, methanethiol; DMDS, dimethyl disulfide; DMTS, dimethyl trisulfide.

FIG. 6.

Proposed transsulfuration pathway and pathways for the formation of VSC in L. casei. metC and malY have been shown to encode the illustrated activities. The degradation of cystathionine to α-ketobutyrate in several Lactobacillus genotypes could not be explained solely by the activity of metC, which is why it is in parentheses. The functions of other genes have been identified by homology (22). metC, cystathionine γ-synthase (with cystathionine γ- and β-lyase activities); malY, CBL (and probably maltose regulon repressor activities); cysK, cysteine synthase (and putative cystathionine synthase), luxS, S-ribosylhomocysteinase; metE, methionine synthase II (cobalamin independent); metK, S-adenosylmethionine synthetase; smtA, S-adenosylmethionine-dependent methyltransferase; pfs, S-adenosylhomocysteine nucleosidase.