PepN is the major aminopeptidase in Escherichia coli:: insights on substrate specificity and role during sodium-salicylate-induced stress
PepN is the major aminopeptidase in Escherichia coli:: insights on substrate specificity and role during sodium-salicylate-induced stress
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DOI：
10.1099/mic.0.26518-0
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发表时间：
2003-12-01
期刊：
影响因子：
2.8
通讯作者：
Nandi, D
中科院分区：
文献类型：
--
作者：
Chandu, D;Nandi, D
在胞质蛋白降解过程中，PepN及其同源物参与了atp无关的步骤（下游加工）。为了深入了解PepN对大肠杆菌肽酶活性的贡献，研究了野生型菌株和两个PepN突变体9218和DH5alphaDeltapepN的提取物中精选的内肽酶和外肽酶底物的水解作用。在两种pepN突变体中，7种内肽酶底物中有3种的水解活性降低。类似的研究表明，在两种pepN突变体中，14种外肽酶底物中有10种的水解程度大大降低。这种切割这些底物的能力下降是pepn特异性的，因为在缺乏其他肽酶，pepA， peb或pepE的大肠杆菌突变体中，水解外肽酶底物的能力没有降低。PepN过表达补充了受影响的外肽酶底物的水解。这些结果表明，PepN是大肠杆菌中大部分氨基肽酶活性的原因。进一步的体外研究表明，纯化的PepN倾向于裂解碱性和小氨基酸作为氨基肽酶底物。动力学表征表明，大肠杆菌PepN的氨基肽酶裂解偏好为Arg b> Ala > Lys > Gly。最后，研究表明，PepN是水杨酸钠诱导的大肠杆菌应激的负调节因子，表明在某些应激条件下，这种氨基内肽酶具有生理作用。
PepN and its homologues are involved in the ATP-independent steps (downstream processing) during cytosolic protein degradation. To obtain insights into the contribution of PepN to the peptidase activity in Escherichia coli, the hydrolysis of a selection of endopeptidase and exopeptidase substrates was studied in extracts of wild-type strains and two pepN mutants, 9218 and DH5alphaDeltapepN. Hydrolysis of three of the seven endopeptidase substrates tested was reduced in both pepN mutants. Similar studies revealed that hydrolysis of 10 of 14 exopeptidase substrates studied was greatly reduced in both pepN mutants. This decreased ability to cleave these substrates is pepN-specific as there is no reduction in the ability to hydrolyse exopeptidase substrates in E coli mutants lacking other peptidases, pepA, pepB or pepE. PepN overexpression complemented the hydrolysis of the affected exopeptidase substrates. These results suggest that PepN is responsible for the majority of aminopeptidase activity in E coli. Further in vitro studies with purified PepN revealed a preference to cleave basic and small amino acids as aminopeptidase substrates. Kinetic characterization revealed the aminopeptidase cleavage preference of E. coli PepN to be Arg > Ala > Lys > Gly. Finally, it was shown that PepN is a negative regulator of the sodium-salicylate-induced stress in E coli, demonstrating a physiological role for this aminoendopeptidase under some stress conditions.