YtjE from Lactococcus lactis IL1403 Is a C-S Lyase with α,γ-Elimination Activity toward Methionine

Sulfur-containing aroma compounds are key contributors to the flavor of a diverse range of foods and beverages. The tropical fruit characters of Vitis vinifera L. cv. Sauvignon blanc wines are attributed to the presence of the aromatic thiols 3-mercaptohexan-1-ol (3MH), 3-mercaptohexan-1-ol-acetate, and 4-mercapto-4-methylpentan-2-one (4MMP). These volatile thiols are found in small amounts in grape juice and are formed from nonvolatile cysteinylated precursors during fermentation. In this study, we overexpressed a Saccharomyces cerevisiae gene, STR3, which led to an increase in 3MH release during fermentation of a V. vinifera L. cv. Sauvignon blanc juice. Characterization of the enzymatic properties of Str3p confirmed it to be a pyridoxal-5-phosphate-dependent cystathionine ␤-lyase, and we demonstrated that this enzyme was able to cleave the cysteinylated precursors of 3MH and 4MMP to release the free thiols. These data provide direct evidence for a yeast enzyme able to release aromatic thiols in vitro that can be applied in the development of self-cloned yeast to enhance wine flavor.

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Engineering Saccharomyces cerevisiae To Release 3-Mercaptohexan-1-ol during Fermentation through Overexpression of an S. cerevisiae Gene, STR3 , for Improvement of Wine Aroma

Holt

Cordente

Williams

et al. 2011

“…The same activities were found for PatC (L. delbrueckii subsp. bulgaricus), PatB (Bacillus subtilis), Lcd (Streptococcus anginosus), and YtjE (L. lactis), which show similarity to MalY (4,5,23,33). Interestingly, overexpression of malY, patB, and patC in CBL-deficient mutants abolished the methionine requirement, showing that they encode CBL activity in vivo.…”

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confidence: 99%

Cloning and Characterization of Two Lactobacillus casei Genes Encoding a Cystathionine Lyase

Irmler

Raboud

Beisert

et al. 2008

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 ␤-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 ␣-keto acid, hydrogen sulfide, or methanethiol. The two enzymes showed notable differences in substrate specificity and pH optimum.Volatile sulfur compounds (VSC) are found in many cheese varieties and are important components of flavor (10). The sulfur compounds methanethiol, methional, and dimethyl trisulfide (DMTS) are key odorants of Swiss Gruyère cheese flavor and are presumably derived from the bacterial metabolism of methionine, which is present in caseins at higher concentrations than cysteine. Swiss Gruyère (place of designated origin) cheese is produced with thermophilic mixed-strain starters which usually contain Streptococcus thermophilus and Lactobacillus delbrueckii subsp. lactis. In addition, depending on the cheese factory, Lactobacillus helveticus can also be present. However, after ripening, Lactobacillus casei and Lactobacillus rhamnosus bacteria are found in high numbers (8). Interestingly, L. casei is also a constituent of the nonstarter lactic acid bacteria of many other cheese varieties (6). Therefore, this species is considered to play an important role during ripening and may be a key player in the formation of flavor compounds.The catabolic pathway(s) of methionine and cysteine in lactobacilli is not well characterized. Possible pathways involve transaminases, methionine ␥-lyases, cystathionine ␤-lyases (CBL), and cystathionine ␥-lyases (CGL) (13). The transamination of methionine by aminotransferases has been studied in lactococci (18,27,34). The results of these studies suggested that methionine is converted to 2-oxo-4-methylthiobutyric acid, which is then converted, either enzymatically or chemically, to methanethiol. In Brevibacterium linens, a methionine ␥-lyases which catalyzes the deamination of methionine and releases methanethiol has been identified and the corresponding gene has been cloned (2,14).CBL, which is encoded by the gene metC in Escherichia coli, is involved in the ␣,␤-elimination of cystathionine to form homocysteine, pyruvate, and ammonia. In comparison, CGL catalyzes the ␣,␥-elimination reaction of cystathionine, producing cysteine, ammonia, and ␣-ketobutyrate. CBL and CGL generally show catalytic promiscuity, which means that they catalyze different reactions. Thus, the CBL from Lactococcus lactis subsp. cremor...

“…Moreover, disruption of the mgl gene encoding the enzyme has been shown to almost eliminate this strain's considerable capacity to produce VSCs (2). In lactococci, which produce only limited amounts of VSCs (15), this reaction is catalyzed by cystathionine lyases (␤-or ␥-) which are responsible for the simultaneous deamination and demethylthiolation of Met to MTL (1,11,18,22); recently, a new C-S lyase (YtjE) with ␣,␥-elimination activity that degrades Met into MTL has been characterized in our laboratory (30). Unfortunately, C-S lyases display relatively low activities toward Met, limiting their capacities to produce VSCs.…”

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confidence: 99%

Heterologous Production of Methionine-γ-Lyase from Brevibacterium linens in Lactococcus lactis and Formation of Volatile Sulfur Compounds

Hanniffy

Philo

Peláez

et al. 2009

Self Cite

The conversion of methionine to volatile sulfur compounds (VSCs) is of great importance in flavor formation during cheese ripening and is the focus of biotechnological approaches toward flavor improvement. A synthetic mgl gene encoding methionine-␥-lyase (MGL) from Brevibacterium linens BL2 was cloned into a Lactococcus lactis expression plasmid under the control of the nisin-inducible promoter PnisA. When expressed in L. lactis and purified as a recombinant protein, MGL was shown to degrade L-methionine as well as other sulfurcontaining compounds such as L-cysteine, L-cystathionine, and L-cystine. Overproduction of MGL in recombinant L. lactis also resulted in an increase in the degradation of these compounds compared to the wild-type strain. Importantly, gas chromatography-mass spectrometry analysis identified considerably higher formation of methanethiol (and its oxidized derivatives dimethyl disulfide and dimethyl trisulfide) in reactions containing either purified protein, whole cells, or cell extracts from the heterologous L. lactis strain. This is the first report of production of MGL from B. linens in L. lactis. Given their significance in cheese flavor development, the use of lactic acid bacteria with enhanced VSC-producing abilities could be an efficient way to enhance cheese flavor development.