br Experimental section br Results and

Experimental section
Results and discussion
Conclusion
Acknowledgement This work was supported by the National Natural Science Foundation of China (Grant Nos. 81401489), the Shanghai Pujiang Program (17PJ1402800) and the Shanghai Sailing Program (Grant No. 14YF1409000).
Introduction Aminopeptidases are ubiquitous housekeeping enzymes found in many microorganisms, plants and animals. They play a crucial role in protein maturation and turnover, catabolism of endogenous and exogenous proteins, and antigen processing [1], [2]. In bacteria and protozoa, the primary role of broad-specificity aminopeptidases is in nutrient acquisition, where they liberate N-terminal SB 216763 from peptides taken up by the cell from the environment [1], [3]. In addition, aminopeptidases found in microbial pathogens have been shown to mediate degradation of host glutathione [4] and adherence to host cells [5], activate bacterial protoxins [6], contribute to biofilm formation [7] and regulate virulence genes [8], suggesting their importance for survival in the host and in pathogenesis. Two aminopeptidases from Plasmodium falciparum have been shown to be essential for parasite viability and validated as therapeutic targets [9]. Aminopeptidases from bacterial pathogens are also being evaluated as targets for drug design [10], [11]. Since aminopeptidases are present in all domains of life, a detailed understanding of the differences in specificity of host and pathogen aminopeptidases must underpin such drug development efforts. The carcinogenic gastric bacterium Helicobacter pylori has two different aminopeptidases. One of these is homologous to methionine aminopeptidases that belong to the M24A peptidase family [12]. It has the putative function of removing the N-terminal methionine from nascent proteins. The second aminopeptidase of H. pylori [hereafter referred to as HpM17AP; previously annotated as peptidase A (PepA) or leucine aminopeptidase (LAP)] contains the signature peptide NTDAEGRL characteristic of M17 aminopeptidases [13] and shows low level overall sequence similarity to other members of this family, with, for example, only 26 and 24% amino-acid sequence identity with the M17 aminopeptidases from Escherichia coli and human, respectively [14]. It is highly induced during nitric oxide (NO) stress [15], [16] and therefore likely plays a role in H. pylori defence against NO generated by macrophages as part of the human innate immune response. In addition, it has been shown that HpM17AP is upregulated in the metronidazole-resistant strain of H. pylori, which suggests that, in addition to having an important housekeeping role, this enzyme contributes to the mechanism of drug resistance [17]. Previous kinetic studies on HpM17AP reported sigmoid rather than hyperbolic velocity versus substrate-concentration plots, indicating that it is an allosteric enzyme [14]. This clearly distinguishes HpM17AP from most of the other characterized M17 aminopeptidases, which show Michaelis–Menten saturation kinetics [18]. M17 aminopeptidases generally have preference for leucine at the N-terminus and have therefore often been termed leucine aminopeptidases (LAPs). However, they also catalyze the hydrolytic removal of other N-terminal amino acids (e.g. Arg, Ala, Trp, Met, Lys [19], [20]). M17 LAPs are hexameric and bind two catalytic metal ions per subunit, which are coordinated by three aspartates, one lysine and one glutamate [2], [13], [21], [22]. Both metal-binding sites must be occupied for an enzyme to be fully active [13], [23]. One of the two sites (site 1) is readily exchangeable, and can be occupied by Zn2+, Mg2+, Mn2+ or Co2+ without any significant difference in catalytic activity. The second site (site 2) is occupied by Zn2+ in most of the characterized LAPs. Zinc binds to site 2 stronger than any metal to site 1, and is retained under conditions that allow exchange of site-1 metal ions [23], [24], [25]. Both ions participate in binding of the substrate and catalytic water molecule and in the stabilization of the transition state intermediate [26]. In addition, the active site of LAPs contains a bicarbonate anion that is bound near the two catalytic metal ions. The bicarbonate ion acts as a general acid/base in the reaction. In the first reaction step, it abstracts a proton from the metal-bridging water molecule, converting it into OH– (stronger nucleophile). The hydroxide ion then makes nucleophilic attack on the carbon atom of the scissile peptide bond to form a tetrahedral gem-diolate reaction intermediate. The site-1 metal ion and a nearby lysine side chain stabilize the oxyanion negative charge in this intermediate [27]. In the second step, the bicarbonate ion acts as an acid by donating the proton to the nitrogen atom, which cleaves the amide bond [28].