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Genetic studies on protein secretion mechanisms of the hyperthermophilic archaea

超嗜热古菌蛋白质分泌机制的遗传学研究

基本信息

批准号：
18608002
负责人：
MATSUMI Rie
金额：
$ 2.71万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

项目摘要

We identified and characterized the signal peptide peptidase (SppA) from the Thermococcus kodakaraensis. In a search for additional enzymes involved in signal peptide degradation, a gene encoding a putative membrane-bound peptidase with similarity to SppA was found. Western blot analysis indicated that the protein (SppB) is membrane-associated. In contrast to SppA, SppB prefers basic amino acids at the P-2 site and hydrophobic residues at the P-1 position. As archaeal signal peptides are comprised from an n-domain (with basic residues) and an h-domain (with hydrophobic residues), the substrate specificities of SppA and SppB can be expected to efficiently complement one another in the initial breakdown of signal peptides at the cytoplasmic membrane. A detailed site-directed mutagenesis study indicated that the peptidase activity of SppB_<Tk> is dependent on a Ser130-His226-Asp154 catalytic triad, distinct to the Ser-Lys catalytic dyad of SppA.We developed a gene disruption system in T. kodakaraensis based on antibiotic resistance. With this system, we disrupted the amylopullulanase gene (apu) or a gene cluster which includes apu and genes of a putative sugar transporter. Cells without the sugar transporter genes could not grow on maltooligosaccharides or polysaccharides, indicating that the gene cluster encodes the only sugar transporter involved in the uptake of these compounds. The Δapu strain could not grow on pullulan and displayed only low levels of growth on amylase, suggesting that Apu is a major polysaccharide-degrading enzyme in T. kodakaraensis.

我们确定和表征信号肽肽酶（SppA）从热球菌kodakaraensis。在寻找参与信号肽降解的其他酶时，发现了一个编码与SppA相似的推定膜结合肽酶的基因，Western印迹分析表明该蛋白（SppB）是膜结合的。与SppA相反，SppB更喜欢P-2位点的碱性氨基酸和P-1位点的疏水残基。由于古细菌信号肽由n-结构域（具有碱性残基）和h-结构域（具有疏水残基）组成，因此可以预期SppA和SppB的底物特异性在细胞质膜上信号肽的初始分解中有效地相互补充。详细的定点突变研究表明，SppB_的肽酶活性<Tk>依赖于一个Ser 130-His 226-Asp 154催化三联体，与SppA的Ser-Lys催化三联体不同。基于抗生素耐药性的柯达卡拉菌。利用该系统，我们破坏了淀粉普鲁兰酶基因（apu）或包含apu和假定的糖转运蛋白基因的基因簇。没有糖转运蛋白基因的细胞不能在麦芽寡糖或多糖上生长，这表明基因簇编码参与摄取这些化合物的唯一糖转运蛋白。Δapu菌株不能在普鲁兰多糖上生长，在淀粉酶上仅显示低水平的生长，表明Apu是T.科达卡拉