PepN, the Major Suc-LLVY-AMC-hydrolyzing Enzyme inEscherichia coli, Displays Functional Similarity with Downstream Processing Enzymes in Archaea and Eukarya

Abstract

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 aminopeptidasespecific 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.Dynamic changes in the proteome of a cell depend on rates of protein synthesis and their degradation. The past few decades have witnessed enormous strides in identifying the molecules and understanding the mechanisms involved in intracellular protein degradation. There is increasing evidence of the involvement of protein degradation in diverse biological activities, e.g. cell cycle progression, transcriptional activation, antigen processing, disease progression, etc. (1-5).Broadly, cytosolic protein degradation is categorized into four steps. (i) Proteins targeted for degradation are initially unfolded into polypeptides by ATP-dependent proteases belonging to the Lon/Clp family in bacteria or 26 S proteasomes in higher organisms (1-5). (ii) These enzymes also make the initial endoproteolytic "cuts" in the polypeptide. Interestingly, in both Escherichia coli (6) and higher organisms (7) the average length of peptides released by these enzymes range from 3 to 25 amino acids. (iii) These longer peptides are trimmed into smaller peptides (less than 10 amino acids) by the action of endopeptidases (8 -11), tripeptidyl-and dipeptidylpeptidases...

Peptidase Activity of the Escherichia coli Hsp31 Chaperone

“…This suggests that Hsp31 is a broad specificity aminopeptidase cleaving after alanine and basic amino acids. Kinetic characterization revealed that Hsp31 displays higher (24) or as the endopeptidase activity of PepN (16,17), but are much lower than the aminopeptidase activity of PepN, the major aminopeptidase of E. coli (16,17).…”

“…The specific aminopeptidase activity of Hsp31 (2120 nmol/h/mg of protein), which is of the same order of magnitude as PepD (1260 nmol/h/mg of protein (24)), is considerably lower than that of PepN (2 ϫ 10 7 nmol/h/mg of protein), the major E. coli aminopeptidase (16,17). We have shown that the aminopeptidase activities assigned to Hsp31 are not contaminated by PepN by extensive purification of Hsp31 and by a different inhibitor sensitivity of Hsp31 and PepN; in contrast with Hsp31, PepN is not inhibited by EDTA or iodoacetamide but is, however, inhibited by PMSF (16 -17).…”

Structure of aminopeptidase N from Escherichia coli suggests a compartmentalized, gated active site

“…Although bacterial PepNs were initially identified as Ala aminopeptidases, subsequent studies demonstrated that Escherichia coli, Lactococcus lactis, and Streptococcus thermophilus PepNs have the highest specificities for the basic amino acids Arg and Lys, followed by hydrophobic͞uncharged residues, like Ala, Leu, Met, and Phe (4, 10). There are conflicting reports as to whether PepN has endopeptidase as well as exopeptidase activity (3,9,11). Based on biochemical studies, Chandu et al (3) have predicted two distinctive active sites, one for the well established exopeptidase activity and a second for the presumed endopeptidase activity of E. coli PepN (ePepN).…”

“…Large complexes, such as Tricorn in archaea, further process these into peptides of 2-3 aa. Finally, various aminopeptidases and carboxypeptidases convert these into individual amino acids (3)(4)(5).…”

“…Aminopeptidase N (PepN) (3,4,6,7) is a zinc-dependent M1-class metalloenzyme classified as a gluzincin based on the conserved active site sequence (HEXXH-X 18 -E) (3,4,8). PepN is the major aminopeptidase in bacteria and accounts for 99% of the hydrolysis of substrates with Ala at the N terminus (9).…”