Biochemical Properties of a Putative Signal Peptide Peptidase from the Hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1
Biochemical Properties of a Putative Signal Peptide Peptidase from the Hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1
复制标题
DOI：
10.1128/jb.187.20.7072-7080.2005
复制
发表时间：
2005-10
影响因子：
3.2
通讯作者：
R. Matsumi;H. Atomi;T. Imanaka
中科院分区：
文献类型：
--
作者：
R. Matsumi;H. Atomi;T. Imanaka
我们首次对来自超嗜热古球菌kodakaraensis KOD1的推定古细菌信号肽肽酶（SppATk）进行了生化表征。SppATk由334个残基组成，比大肠杆菌的对应物（618个残基）要小得多，并且在其N端附近有一个预测的跨膜结构域。一个没有n端54个氨基酸残基的截断突变蛋白（ΔN54SppATk）被发现对自身蛋白水解是稳定的，并进行了进一步的研究。ΔN54SppATk对荧光肽底物表现出肽酶活性，并被发现具有高度的耐热性。此外，该酶表现出显著的稳定性和对碱性的偏好，在pH 10时活性最高。ΔN54SppATk在80℃、pH 10条件下对ala - ala - pH -4-甲基-香豆醇-7-酰胺的反应Km为240±18 μM， Vmax为27.8±0.7 μmol min−1 mg−1。该酶的底物特异性用FRETS肽库进行了详细的检测。通过液相色谱-质谱分析裂解产物，发现ΔN54SppATk能有效地裂解P-1位侧链相对较小，P-3位有疏水或芳香残基的肽。带正电的Arg残基优先位于P-4位点，而带负电残基位于P-2、P-3或P-4位点的底物不会被切割。当对T. kodakaraensis基因组序列的预测信号序列进行检测时，我们发现ΔN54SppATk的底物特异性与其在该古菌中作为信号肽酶的假设作用非常一致。
ABSTRACT We have performed the first biochemical characterization of a putative archaeal signal peptide peptidase (SppATk) from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. SppATk, comprised of 334 residues, was much smaller than its counterpart from Escherichia coli (618 residues) and harbored a single predicted transmembrane domain near its N terminus. A truncated mutant protein without the N-terminal 54 amino acid residues (ΔN54SppATk) was found to be stable against autoproteolysis and was examined further. ΔN54SppATk exhibited peptidase activity towards fluorogenic peptide substrates and was found to be highly thermostable. Moreover, the enzyme displayed a remarkable stability and preference for alkaline pH, with optimal activity detected at pH 10. ΔN54SppATk displayed a Km of 240 ± 18 μM and a Vmax of 27.8 ± 0.7 μmol min−1 mg−1 towards Ala-Ala-Phe-4-methyl-coumaryl-7-amide at 80°C and pH 10. The substrate specificity of the enzyme was examined in detail with a FRETS peptide library. By analyzing the cleavage products with liquid chromatography-mass spectrometry, ΔN54SppATk was found to efficiently cleave peptides with a relatively small side chain at the P-1 position and a hydrophobic or aromatic residue at the P-3 position. The positively charged Arg residue was preferred at the P-4 position, while substrates with negatively charged residues at the P-2, P-3, or P-4 position were not cleaved. When predicted signal sequences from the T. kodakaraensis genome sequence were examined, we found that the substrate specificity of ΔN54SppATk was in good agreement with its presumed role as a signal peptide peptidase in this archaeon.