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  • Analysis of the crystal structures of LAPs from E

    2024-02-19

    Analysis of the crystal structures of LAPs from E. coli[29], bovine eye lens [30], Pseudomonas putida[27], tomato [31] and P. falciparum[32] revealed that the monomer of M17-family LAP is composed of two domains: the smaller, variable N-terminal domain and the larger, conserved C-terminal domain that contains the active site. In the context of the biological hexamer, the six active sites of LAP are located in the cavity in the interior of the oligomer, and each substrate-binding channel can accommodate peptides of up to five residues in length [31], which is consistent with LAP's preference for short peptides [33]. M17 LAPs are reversibly inhibited by bestatin, a natural pseudopeptide (Fig. 1). Its chemical structure resembles that of the dipeptide Phe-Leu, but contains α-hydroxy-β-phenylalanine at the N-terminus instead of phenylalanine. In addition, the chiral C3 carbon atom, to which the phenylalanine side chain and the terminal amino group are attached, has an R-configuration whereas the equivalent carbon induced pluripotent stem cells of a natural peptide substrate has an S-configuration [27]. A previous X-ray crystallographic study of LAP from bovine eye lens showed that bestatin functions as a competitive inhibitor by binding in the enzyme's active site, with the D-phenylalanyl and L-leucyl side chains occupying the S1 and S1' subsites, respectively [30], [34]. In common with other M17 aminopeptidases, HpM17AP activity is inhibited by bestatin [14]. Moreover, previous studies have shown that bestatin inhibits H. pylori growth [14]. Here, we report the crystal structures of HpM17AP and its bestatin complex determined at 2.0 Å and 1.9 Å resolution, respectively. In addition, we present an investigation of the preferences of HpM17AP for residues in peptide substrates. Together, this analysis allows us to address the structural basis of substrate specificity and catalysis of this enzyme.
    Results
    Discussion Due to their role in survival in the host and in pathogenesis, microbial M17 aminopeptidases have gained much attention in recent years. This study adds to the growing evidence that within this family, there are enzymes that do not use N-terminal leucine as the preferred substrate, and therefore the original generic name ‘leucine aminopeptidase (LAP)’ has become obsolete and further subdivision into sub-families on the basis of substrate specificity would be appropriate. Profiling the substrate specificity revealed that like SaM17AP, HpM17AP displays a strong preference for L-Arg over L-Leu. The crystal structure of HpM17AP identified elements that are shared by the H. pylori and S. aureus enzymes, but are absent in their homologues that have preference towards leucine. Analysis of the structural differences between HpM17AP and SaM17AP on the one hand, and PpLAP and EcLAP on the other, appears to be fully consistent with differences in their specificity and pinpoints key structural features that might contribute to specificity of these two sub-families. H. pylori cannot synthesize its own L-arginine and therefore must obtain it from the environment within the host [40]. H. pylori contains genes for dipeptide and oligopeptide transporters [12] which allow it to import peptides from the gastric mucosa. High activity of HpM17AP on peptides with N-terminal L-arginine may be important for maintaining a sufficient cytoplasmic pool of free L-arginine, which in turn could be used for synthesis of polyamines that are needed for optimal growth of H. pylori. Our observation of significant activity of HpM17AP on N-terminal L-cysteine is particularly interesting in the view of the recent reports linking the cysteine-glycinase activity of M17 aminopeptidases from pathogenic bacteria to glutathione metabolism [4], [20], [41]. E. coli, Francisella tularensis and Treponema denticola can utilize exogenous glutathione as a cysteine source in a γ-glutamyl transpeptidase- and cysteine-glycinase-dependent manner [4], [41], [42]. Similarly, H. pylori has been shown to utilize the stomach's mucosal glutathione, which is produced as the major defense mechanism against low pH, oxidative and osmotic stress [43], as a source of glutamate [44]. The resultant Cys-Gly dipeptide produced by the action of H. pylori γ-glutamyl transpeptidase is likely imported into the bacterial cell via one of its several dipeptide transporters and cleaved by as yet unidentified aminopeptidase to salvage cysteine. In future studies, it would therefore be important to establish whether HpM17AP uses Cys-Gly as a physiological substrate and what role this may play in the pathogenesis of H. pylori infection.