PepN, the major Suc-LLVY-AMC-hydrolyzing enzyme in Escherichia coli, displays functional similarity with downstream processing enzymes in archaea and eukarya -: Implications in cytosolic protein degradation
PepN, the major Suc-LLVY-AMC-hydrolyzing enzyme in Escherichia coli, displays functional similarity with downstream processing enzymes in archaea and eukarya -: Implications in cytosolic protein degradation
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DOI：
10.1074/jbc.m207926200
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发表时间：
2003-02-21
影响因子：
4.8
通讯作者：
Nandi, D
中科院分区：
文献类型：
--
作者：
Chandu, D;Kumar, A;Nandi, D
Succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin(Suc-LLVY-AMC)是一种荧光内肽酶底物，用来检测从古生物到哺乳动物的20种S蛋白酶体的活性。在缺少20个S蛋白酶体的大肠杆菌中仍检测到邻菲咯啉敏感的Suc-LLVY-AMC水解酶活性。我们确定PepN负责Suc-LLVY-AMC的水解酶，该酶以前被鉴定为大肠杆菌中唯一的丙氨酸氨基肽酶。PepN是一种氨基内肽酶。首先，突变体9218的提取物不裂解Suc-LLVY-AMC和L-丙氨酸-对硝基苯胺(PNA)。其次，生化纯化的PepN可同时切割多种氨基肽酶和内肽酶底物，其中L-丙氨酸-PNA的切割效率高于其他底物。对氨肽酶特异性抑制剂Bestatin的研究表明，氨基肽酶和内肽酶底物的裂解机制不同。第三，PepN能分解整个蛋白质、酪蛋白和白蛋白。最后，一株具有PepN靶向缺失的大肠杆菌菌株也缺乏切割Suc-LLVY-AMC和L-Ala-PNA的能力，而野生型PepN在该突变体中的表达挽救了这两种活性。此外，我们还通过质谱分析，在含有20个S蛋白酶体的真细菌污垢分枝杆菌中鉴定出一个低分子量的Suc-LLVY-AMC裂解酶，它是一种与E.ColiPepN同源的氨基肽酶。PepN的“基于序列的同源物”包括特征明确的氨基肽酶，如古生代的三角兽相互作用因子F2和F3，以及哺乳动物中的嘌呤霉素敏感的氨基肽酶。然而，我们的结果表明，显示氨基内肽酶活性的真细菌PepN及其同系物可能在功能上类似于胞浆中蛋白质下游加工中重要的酶：在古生物中是tricorn-F1-F2-F3复合体，在真核生物中是TPPII/Multicorn。
Succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin (Suc-LLVY-AMC), a fluorogenic endopeptidase substrate, is used to detect 20 S proteasomal activity from Archaea to mammals. An o-phenanthroline-sensitive Suc-LLVY-AMC hydrolyzing activity was detected in Escherichia coli although it lacks 20 S proteasomes. We identified PepN, previously characterized as the sole alanine aminopeptidase in E. coli, to be responsible for the hydrolysis of Suc-LLVY-AMC. PepN is an aminoendopeptidase. First, extracts from an ethyl methanesulfonate-derived PepN mutant, 9218, did not cleave Suc-LLVY-AMC and L-Ala-para-nitroanilide (pNA). Second, biochemically purified PepN cleaves a wide variety of both aminopeptidase and endopeptidase substrates, and L-Ala-pNA is cleaved more efficiently than other substrates. Studies with bestatin, an aminopeptidase-specific inhibitor, suggest differences in the mechanisms of cleavage of aminopeptidase and endopeptidase substrates. Third, PepN hydrolyzes whole proteins, casein and albumin. Finally, an E. coli strain with a targeted deletion in PepN also lacks the ability to cleave Suc-LLVY-AMC and L-Ala-pNA, and expression of wild type PepN in this mutant rescues both activities. In addition, we identified a low molecular weight Suc-LLVY-AMC-cleaving peptidase in Mycobacterium smegmatis, a eubacteria harboring 20 S proteasomes, to be an aminopeptidase homologous to E. coli PepN, by mass spectrometry analysis. "Sequence-based homologues" of PepN include well characterized aminopeptidases, e.g. Tricorn interacting factors F2 and F3 in Archaea and puromycin-sensitive aminopeptidase in mammals. However, our results suggest that eubacterial PepN and its homologues displaying aminoendopeptidase activities may be "functionally similar" to enzymes important in downstream processing of proteins in the cytosol: Tricorn-F1-F2-F3 complex in Archaea and TPPII/Multicorn in eukaryotes.