CysK from Lactobacillus casei encodes a protein with O-acetylserine sulfhydrylase and cysteine desulfurization activity

Abstract

A gene encoding an O-acetyl-L-serine sulfhydrylase (cysK) was cloned from Lactobacillus casei FAM18110 and expressed in Escherichia coli. The purified recombinant enzyme synthesized cysteine from sulfide and O-acetyl-L-serine at pH 5.5 and pH 7.4. At pH 7.4, the apparent K(M) for O-acetyl-L-serine (OAS) and sulfide were 0.6 and 6.7 mM, respectively. Furthermore, the enzyme showed cysteine desulfurization activity in the presence of dithiothreitol at pH 7.5, but not at pH 5.5. The apparent K(M) for L-cysteine was 0.7 mM. The synthesis of cystathionine from homocysteine and serine or OAS was not observed. When expressed in a cysMK mutant of Escherichia coli, the cloned gene complemented the cysteine auxotrophy of the mutant. These findings suggested that the gene product is mainly involved in cysteine biosynthesis in L. casei. Quantitative real-time PCR and a mass spectrometric assay based on selected reaction monitoring demonstrated that L. casei FAM18110 is constitutively overexpressing cysK.

A gene horizontally transferred from bacteria protects arthropods from host plant cyanide poisoning

“…(The ratio was calculated from the respective values of V max /K m , as k cat = V max /[E] and as the reactions were measured with the same enzyme preparation.) This preferred CAS activity is typical of known plant CAS enzymes and clearly different from CYS enzymes (Table 1; Yamaguchi et al, 2000; Wada et al, 2004; Bogicevic et al, 2012; Yi et al, 2012). Enzymatic activity was dependent on pyridoxal-5′-phosphate as a cofactor, and the substrate-dependent formation of β-cyanoalanine was further confirmed by thin layer chromatography (TLC) and LC-MS (Figure 7).…”

“…CAS activity was measured with cysteine as substrate while CYS activity was measured with O -acetylserine as substrate. Data for the plants A. thaliana and G. max were obtained in Yamaguchi et al (2000) and Yi et al (2012), respectively, while data for the bacteria C. glutamaticum and L. casei were retrieved from Wada et al (2004) and Bogicevic et al (2012), respectively.

10.7554/eLife.02365.012Figure 7.Panel A: Formation and accumulation of β-cyanoalanine by recombinant Tu-CAS as visualized by TLC analysis.Controls; C1: no cysteine control, C2: no cyanide control, C3: no enzyme control. Time course 0–60 min after adding 3.75 µg of recombinant Tu-CAS.

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“…CAS activity was measured with cysteine as substrate while CYS activity was measured with O -acetylserine as substrate. Data for the plants A. thaliana and G. max were obtained in Yamaguchi et al (2000) and Yi et al (2012), respectively, while data for the bacteria C. glutamaticum and L. casei were retrieved from Wada et al (2004) and Bogicevic et al (2012), respectively.…”

Functional genomics of Lactobacillus casei establishment in the gut

“…6 and Table S2). The cysteine synthase activity of the enzyme encoded by LSEI_0480 was demonstrated experimentally (36). Because LSEI_0479 and 0480 form an operon (Fig.…”

Coupled carbon, sulfur and nitrogen cycles of mixotrophic growth of Pseudomonas sp. C27 under denitrifying sulfide removal conditions

“…Apart from being oxidized to S 0 , sulfide is also involved in carbon metabolism. Sulfide was reported to react with O-Acetyl-L-serine to form L-cysteine and acetate by cysteine synthase A catalyzing (Kredich and Tomkins, 1996) by Lactobacillus (Bogicevic et al, 2012) or by Escherichia (Uda et al, 2013). Cysteine synthase A was claimed not only catalyzing synthesis of cysteine, but also having activity of desulfurization (Awano et al, 2005;Flint et al, 1996).…”