Intracellular protein degradation is required for maintaining the cellular proteome and regulating cellular processes. This pathway involves proximal ATP-dependent proteases that unfold and translocate proteins targeted for degradation into catalytic chambers. The large peptides produced are further cleaved by ATP independent endopeptidases, aminopeptidases and carboxypeptidases to release free amino acids. Lon and Clp are the key ATP-dependent proteases in prokaryotes and 26S proteasomes in eukayotes. In general, enzymes involved in the distal processing of peptides are ATP-independent, display greater redundancy and their orthologs are present in most organisms. The aim of the present study was to generate biochemical and functional insights on the ATP-independent enzyme, Peptidase N (PepN), which belongs to the M1 family. Previous studies in our laboratory identified Escherichia Coli PepN, to harbor both amino and endopeptidase activitities. In addition, it is responsible for the cleavage of majority of aminopeptidase substrates in E. Coli and is known to be involved in Sodium salicylate(NaSal)-induced stress. The present study consists of four parts. First, intracellular proteolysis plays an important role for virulence in pathogens. Therefore, it becomes important to study the biochemical properties and roles of enzymes involved in protein degradation. In this direction, a study was initiated to characterize the biochemical properties of Peptidase N from Salmonella enterica serovar Typhimurium(S. typhimurium). To study the contribution of PepN to the overall cystosolic protein degradation in S.typhimurium, a targeted deletion in pepN was generated. Cystosolic lysates of S. typhimurium wild type(WT) and ΔpepN strains were examined for their ability to cleave a panel of aminopeptidase and endopeptidase substrates. The ΔpepN strain displayed greatly reduced cleavage of nine out of a total of thirteen exopeptidase substrates, demonstrating a significant contribution of PepN to cytosolic aminopeptidase activity. S. typhimurium PepN also cleaved the endopeptidase substrate Suc-LLVY-AMC, similar to E. Coli PepN. To understand the physiological role of PepN, WT and ΔpepN were subjected to different stress conditions. During nutritional downshift in combination with high temperature stress, the growth of ΔpepN was significantly reduced compared to WT. Importantly, the PepN overexpressing strains grew better than WT, demonstrating an enhanced ability to overcome this stress combination. The above study clearly underscores the importance of PepN, to play distinct roles during stress. The significance of this study lies in understanding the biochemical and functional properties of a M1 family member from a pathogenic organism. Second, peptidases belonging to the M1 family are widely distributed with orthologs found across different kingdoms. The key amino acids in the catalytic domain are conserved in this family. However, amino acids present in the C-termini are variable and the three available crystal structures of M1 family members display distint differences in organization of this domain. To investigate the functional role of C-termini, progressive deletions were generated in PepN from E.Coli and Tricorn interacting factor F2 from Thermoplasma acidophilum(F2). Catalytic activity was partially reduced inPepN lacking four aa from C-terminus (PepNΔC4) whereas it is greatly reduced in F2 lacking ten amino acids from C-terminus(F2ΔC10) or eleven amino acids from PepN (PepNΔC11). To understand the mechanistic reasons involved, biochemical and biophysical studies were performed on purified WT and C-termini deleted proteins. Increased binding to 8-amino- 1- naphthalene sulphonic acid (ANS) was observed for all C-termini deleted proteins revealing greater numbers of surface exposed hydrophobic amino acids. Further, trypsin sensitivity studies demonstrated that mutant proteins were more sensitive compared to WT. Notably, expression of PepNΔC4, but not PepNΔC11, in E ColiΔpepN increased its ability to resist nutritional and high temperature stress, demonstrating a physiological role for the C-terminus. Together, these studies reveal involvement of distal amino acids in the C-termini of two distant M1 family members in repressing the exposure of apolar residues and enhancing enzyme function. Third, the crystal structure of E. coliPepN displayed the presence of Zn2+. To study the role of metal cofactor, apo-PepN was isolated by chelating the holoenzyme with 1,10-phenanthroline. Among different metals tested, only Zn2+ rescued the greatly reduced catalytic activity of the apo-PepN. Further confirmatory studies were performed using pepN mutants in the conserved GXMEN and HEXXH motifs. No major structural differences were observed in purified mutants(E264A, H297A, and E298A) using circular dichroism (CD) and intrinsic fluorescence studies; however, they lacked catalytic activity. These studies clearly demonstrate that Zn2+ was essential for catalysis but not for the overall structural integrity of PepN. Estimation of the Zn2+ content by atomic absorption spectrometry demonstrated that the WT contained one molecule of zinc per molecule of enzyme. Similar results were obtained in purified proteins of E264A and E298A. residues involved in catalysis. However the Zn2+ amount was greatly reduced in H297A, which is involved in Zn2+ binding. Further, the in vivo role of metal cofactor and catalyis were studied during two established stress conditions. Over expression of the mutants, unlike WT, was unable to rescue the growth of ΔpepN during nutritional down shift and high temperature stress. These results demonstrate that E264, H297 and E298 were required for PepN function during nutritional downshift and high temperature stress. However during NaSal-induced stress condition, overexpression of WT or mutants reduced growth of ΔpepN, demonstrating that PepN function was independent of catalytic activity or metal cofactor. Further studies identified the YL motif, which is conserved in all members of the M1 family, to play a role during NaSal-induced stress. Over expression of Y185F or L186Q did not modulate catalytic activity although growth reduction of ΔpepN in the presence of NaSal was compromised. To understand the mechanisms by which the YL motif plays a role during this condition, Y185F and L186Q mutant proteins were purified. In vitro, both mutant proteins were found to aggregate at a lower temperature and their catalytic activities were more sensitive to temperature, compared to WT. Steady state analysis of WT, Y185F and L186Q were performed to study the modulation of PepN amount during stress conditions. Steady state amounts of Y185F and L186Q mutant proteins were greatly decreased compared to WT, during NaSal-induced stress. Most likely, the lowered amounts of Y185F and L186Q mutant proteins contribute to growth advantage during NaSal-induced stress. Thus, the YL motif in E. Coli PepN reduces protein aggregation and enhances the structural integrity of PepN during selective stress conditions in vivo. In summary, this study clearly identifies metal cofactor and peptidase-dependent and –independent motifs to play distinct functional roles in PepN. Fourth, the crystal structures of known M1 family members have shown that the catalytic domain and mechanism of action are similar. To identify novel residues that may modulate the catalytic activity of PepN, multiple sequence alignment of important M1 family members were performed. The alignment identified a subset of M1 family members, including PepN, containing an aspargine residue which is present two amino acids before glycine in the GAMEN motif. A closer investigation of thecrystal structure of PepN revealed an interaction between N259(Catalytic domain) with Q821 (C-terminal domain). To understand the functional role of this interaction, site-specific mutants were generated: N259D, Q821E and a double mutant, N259D & Q821E. Spectroscopic studies did not reveal any significant differences with respect to global structure or protein stability between purified WT and mutant enzymes. Also, binding to substrates by mutant enzymes was not affected as judged by Km values. However, the Kcat of PepN containing N259D or Q821E was enhanced with respect to both aminopeptidase and endopeptidase substrates. On the other hand, there was significant decrease in the catalytic activity of the double mutant. Modeling studies demonstrate that the N259-Q821 interaction is located in the vicinity of residues important for catalysis in PepN and specific alterations in this interaction may affect the compactness of the catalytic domain. In summary, this study provides a functional role for the N259-Q821 interaction in modulating the catalytic activity of PepN. Mammalian orthologs of M1 family members play important roles in different physiological processes, e.g. angiogenesis, blood pressure, inflammation, MHC class I antigen presentation etc. PepN is a well characterized M1 family member of microbial origin. The present study on E. Coli PepN provides new knowledge on the roles of: a) distal C-terminal amino acids in repressing exposed hydrophobic amino acids; b) the conserved YL motif during NaSal-induced stress condition; c) the N259 and Q821 interaction in modulating enzymatic activity. The implications of these results on other members of the M1 family are discussed.