Heterologous gene expression and characterization of two serine hydroxymethyltransferases from Thermoplasma acidophilum
Heterologous gene expression and characterization of two serine hydroxymethyltransferases from Thermoplasma acidophilum
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DOI：
10.1007/s00792-021-01238-9
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发表时间：
2021-07-01
期刊：
影响因子：
2.9
通讯作者：
Honda, Kohsuke
中科院分区：
文献类型：
--
作者：
Fauziah Ma'ruf, Ilma;Sasaki, Yuka;Honda, Kohsuke
丝氨酸羟甲基转移酶（SHMT）和苏氨酸醛缩酶被归类为折叠I型吡哆醛-5'-磷酸依赖酶，分别参与丝氨酸和苏氨酸的甘氨酸生物合成。嗜酸嗜酸古菌Thermoplasma acidophilum具有两个不同的SHMT基因，而其基因组中没有编码苏氨酸醛缩酶的基因。本研究将两个SHMT基因Ta0811和Ta1509分别在大肠杆菌和kodakarensis热球菌中异种表达，并对其产物的生化特性进行了研究。Ta1509蛋白具有催化四氢叶酸（THF）依赖性丝氨酸裂解和不依赖THF的苏氨酸裂解的双重活性，与迄今报道的其他shmt相似。相反，Ta0811蛋白缺少thf结合基序的氨基酸残基，只催化不依赖thf的苏氨酸裂解。动力学分析表明，Ta0811蛋白的苏氨酸裂解活性比Ta1509蛋白的丝氨酸裂解活性高3.5倍。此外，Ta0811基因在嗜酸乳杆菌中的mRNA表达量约为Ta1509的20倍。这些观察结果表明，由Ta0811蛋白介导的苏氨酸的后醛醇裂解在嗜酸乳杆菌的甘氨酸生物合成中起着重要作用。
Serine hydroxymethyltransferase (SHMT) and threonine aldolase are classified as fold type I pyridoxal-5'-phosphate-dependent enzymes and engaged in glycine biosynthesis from serine and threonine, respectively. The acidothermophilic archaeon Thermoplasma acidophilum possesses two distinct SHMT genes, while there is no gene encoding threonine aldolase in its genome. In the present study, the two SHMT genes (Ta0811 and Ta1509) were heterologously expressed in Escherichia coli and Thermococcus kodakarensis, respectively, and biochemical properties of their products were investigated. Ta1509 protein exhibited dual activities to catalyze tetrahydrofolate (THF)-dependent serine cleavage and THF-independent threonine cleavage, similar to other SHMTs reported to date. In contrast, the Ta0811 protein lacks amino acid residues involved in the THF-binding motif and catalyzes only the THF-independent cleavage of threonine. Kinetic analysis revealed that the threonine-cleavage activity of the Ta0811 protein was 3.5 times higher than the serine-cleavage activity of Ta1509 protein. In addition, mRNA expression of Ta0811 gene in T. acidophilum was approximately 20 times more abundant than that of Ta1509. These observations suggest that retroaldol cleavage of threonine, mediated by the Ta0811 protein, has a major role in glycine biosynthesis in T. acidophilum.