CysK from Lactobacillus casei encodes a protein with O-acetylserine sulfhydrylase and cysteine desulfurization activity
CysK from Lactobacillus casei encodes a protein with O-acetylserine sulfhydrylase and cysteine desulfurization activity
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DOI：
10.1007/s00253-011-3677-5
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发表时间：
2012-06-01
影响因子：
5
通讯作者：
Irmler, Stefan
中科院分区：
文献类型：
--
作者：
Bogicevic, Biljana;Berthoud, Helene;Irmler, Stefan
从干酪乳杆菌FAM 18110中克隆了编码O-乙酰-L-丝氨酸硫化氢解酶（cysK）的基因，并在大肠杆菌中表达。纯化的重组酶在pH5.5和pH7.4下由硫化物和O-乙酰-L-丝氨酸合成半胱氨酸。在pH 7.4时，O-乙酰基-L-丝氨酸（OAS）和硫化物的表观KM分别为0.6和6.7 mM。此外，该酶在pH 7.5的二硫苏糖醇的存在下，但不是在pH 5.5的半胱氨酸脱硫活性。L-半胱氨酸的表观KM为0.7 mM。未观察到由同型半胱氨酸和丝氨酸或OAS合成胱硫醚。当在大肠杆菌的cysMK突变体中表达时，克隆的基因补充突变体的半胱氨酸营养缺陷型。这些结果表明，该基因产物主要参与L. casei。定量实时PCR和质谱分析的基础上选择的反应监测表明，L。casei FAM 18110组成型过表达cysK。
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 KM 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 KM 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.