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

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.