The leucyl aminopeptidase from Helicobacter pylori is an allosteric enzyme

Lei Dong1,{dagger}, Ni Cheng1,{dagger}, Ming-Wei Wang2, Junfeng Zhang1, Chang Shu1 and De-Xu Zhu1

1 State Key Laboratory of Pharmaceutical Biotechnology, Department of Biochemistry, Nanjing University, Nanjing 210093, P. R. China
2 The National Center for Drug Screening, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, P. R. China

Correspondence
De-Xu Zhu
zjq{at}nju.edu.cn


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
This study describes the cloning, genetic analysis and biochemical characterization of a leucyl aminopeptidase (LAP) from Helicobacter pylori. A gene encoding LAP was cloned from H. pylori and the expressed 55 kDa protein displayed homology to aminopeptidases from Gram-negative bacteria, plants and mammals. This LAP demonstrated amidolytic activity against L-leucine-p-nitroanilide. Optimal activity was observed at pH 8·0 and 45 °C, with Vmax of 232 µmol min–1 (mg protein)–1 and S0·5 of 0·65 mM. The data suggest that LAP could be allosteric (nH=2·27), with regulatory homohexamers, and its activity was inhibited by ion chelators and enhanced by divalent manganese, cobalt and nickel cations. Bestatin inhibited both LAP activity (IC50=49·9 nM) and H. pylori growth in vitro. The results point to the potential use of LAP as a drug target to develop novel anti-H. pylori agents.


Abbreviations: ICP-AES, inductively coupled plasma-atomic emission spectrometry; LAP, leucyl aminopeptidase; L-Leu-p-NA, L-leucine-p-nitroanilide

{dagger}These authors contributed equally to this work.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Helicobacter pylori is a microaerophilic, Gram-negative, spiral, flagellated bacterium which causes superficial gastritis, chronic atrophic gastritis, peptic ulcer and gastric cancer (Hopkins et al., 1996; Kreiss et al., 1995). It is prevalent in almost half the world's population (Cover & Blaser, 1996). No vaccine is available at present and anti-microbial therapy for the infection is a complex issue. Although current optimal first-line treatment, consisting of proton-pump inhibitors and/or bismuth, metronidazole, clarithromycin or amoxicillin (Malfertheiner et al., 2002), is associated with high cure rates, the rising incidence of resistance to the antibiotics increasingly threatens to compromise the efficacy of these eradication regimens (Björkholm et al., 2001). Therapeutic agents directed against H. pylori infection will continue to evolve and there is a pressing need for the identification of novel drug targets, such as enzymes vital to the survival of this bacterium. This opportunity exists now as a result of successful sequencing of the genomes of two H. pylori strains (Tomb et al., 1997; Alm et al., 1999).

Aminopeptidases, which catalyse the removal of N-terminal amino acid residues from peptides and proteins (Taylor, 1993), play an important role in several physiological processes. It is noteworthy that some of them take part in the catabolism of exogenously supplied proteins (Smid et al., 1991; Booth et al., 1990), and are necessary for the final steps of protein turnover (Lazdunski, 1989; Goldberg & Dice, 1974; Goldberg & John, 1976) and maturation (Lazdunski, 1989; Miller 1975). Bacterial aminopeptidases can be classified based on their catalytic mechanisms: metallo-, cysteine and serine aminopeptidases. The metalloaminopeptidase is predominant in bacteria; its activity is regulated by the presence of divalent metallic cations and may be inhibited by chelating agents (e.g. EDTA) (Thierry & Janine, 1996). Most of the above studies demonstrate that bacterial aminopeptidases generally show Michaelis–Menten kinetics though they possess a multimeric structure. Very few of them display allosteric kinetics.

Because of their critical role in the life cycle of micro-organisms, aminopeptidases are emerging as novel and promising drug targets, especially in the development of new anti-parasitic agents (Niven, 1991; Nankya-Kitaka et al., 1998). Some aminopeptidase inhibitors show good efficacy against parasites such as Plasmodium falciparium and Trypanosoma brucei (Knowles, 1993; Howarth & Lloyd, 2000). Two other aminopeptidase inhibitors, Bestatin and 1,10-phenanthroline, exhibit notable inhibitory effects on the growth of Fusobacterium nucleatum (Rogers et al., 1998). However, aminopeptidase from H. pylori has not yet been reported.

In this paper, we describe the cloning, genetic analysis and biochemical characterization of a leucyl aminopeptidase (LAP) from a H. pylori standard strain (ATCC 43504). The data presented considerably expand our knowledge, which thus far is rather limited, of the peptidolytic capacity of H. pylori.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacteria and culture conditions.
A standard strain of H. pylori (ATCC 43504) was kept at –80 °C in 10 % sucrose and 50 % heat-inactivated fetal bovine serum (FBS). It was cultured on Columbia agar (Difco) containing 10 % defibrinated sheep blood and then passaged to Brucella broth supplemented with 5 % FBS, as described by Müller et al. (1991). Plates and tubes inoculated with H. pylori were incubated at 37 °C under microaerophilic conditions (10 % CO2, 5 % O2 and 85 % N2) for 1–4 days. Escherichia coli BL21 (DE3) pLysE was grown at 37 °C in Luria broth or agar supplemented with antibiotics when needed.

Cloning, sequencing and analysis of the LAP ORF.
Using the published sequence of the H. pylori genome (GenBank accession number NC_000915; gene HP0570), forward (5'-GAC CAT ATG TTA AAA ATC AAA TTA GAA AAA ACC-3') and reverse primers (5'-CCC CTC GAG AGC CTT TTT CAA AAG CTC TT-3') were synthesized that were complementary to the complete coding sequences flanking a H. pylori ORF designated ‘probable cytosol aminopeptidase’. Built-in NdeI and XhoI sites are shown by bold type and the initiation codon is underlined. The stop codon was built into the plasmid vector. PCR amplification of genomic DNA from H. pylori was carried out in the following order: initial denaturation (95 °C, 2 min), 30 cycles of denaturation (95 °C, 1 min), annealing (50 °C, 1 min), primer extension (72 °C, 2 min) and final extension (72 °C, 10 min). PCR products were cloned into pET26b (Novagen). The plasmid was sequenced using T7 forward primer and M13 reverse primer, and the result indicated that the ATG start codon was in-frame.

Hyperexpression of recombinant LAP.
The LAP gene was cloned into pET26B and the construct was transformed into E. coli pLysE. The N-terminal polyhistidine-tagged fusion protein was expressed by induction of exponential-phase culture (500 ml; OD600~0·6, as determined by a UV2001 spectrophotometer, Shimadzu) with 1 mM IPTG for 4 h at 37 °C with vigorous (300 r.p.m.) shaking. Bacteria were harvested by centrifugation (7000 g, 10 min, 4 °C), resuspended in 20 mM Tris/HCl, 500 mM NaCl and 1 mM PMSF, pH 7·9 (20 ml, 4 °C), and sonicated with a Branson Sonifer 250 sonicator (Branson Ultrasonics Corp.). Centrifuged (15 000 g, 15 min, 4 °C) extracts were heated in 65 °C water for 10 min, centrifuged again (15 000 g, 15 min, 4 °C), and peptidases purified on nickel–agarose resin (Novagen) according to the manufacturer's instructions. Sample purity was evaluated on a Coomassie blue-stained 12 % Tris/Tricine SDS-PAGE gel. A Sephadex G200 column (Amersham Biosciences) was used to determine the molecular mass of the enzyme in its oligomerization state. The total column volume was approximately 60 ml, and the standard buffer was 50 mM Tris/HCl, 100 mM NaCl, 1 mM EDTA, with or without 1 mM DTT, at pH 8·0. The metal content of LAP protein was analysed by inductively coupled plasma-atomic emission spectrometry (ICP-AES) at the Contemporary Analytical Center, Nanjing University.

Enzymic analysis of recombinant LAP.
Kinetic analysis of LAP was carried out according to the procedure of Tan & Konings (1990), with some modifications. The standard reaction mixture contained 50 mM Tris/HCl (pH 8·0), 0·5 µM MnCl2, 15 µg LAP protein and an appropriate amount of L-leucine-p-nitroanilide (L-Leu-p-NA). After incubation at 37 °C for 10 min, the enzyme and L-Leu-p-NA were sequentially added to start the reaction. Absorbance at 405 nm was continuously measured for a minimum of 2 min by UV2001 spectrophotometer (Shimadzu). The initial velocity was calculated from the slope of the linear range of an absorbance versus time curve. Vmax was obtained from the substrate-saturation curve. The Hill plot method (Dixon & Webb, 1979) was used to analyse the data to achieve half-saturation constant (S0·5) and Hill number (nH). S0·5 values are defined as the concentration of the substrate that gives 50 % maximal activity.

The LAP pH profile was determined by incubating the enzyme (15 µg ml–1, 37 °C, 5 min) in constant ionic strength acetate/MES/Tris (AMT) buffer [50 mM acetic acid, 50 mM MES and 100 mM Tris/HCl (Ellis & Morrison, 1982), pH 4–11] and 0·5 mM MnCl2 before addition of L-Leu-p-NA (final concentration 5 mM).

The metal ion dependence of LAP was investigated by assaying LAP activity after preincubation of the enzyme (15 µg ml–1, 37 °C, 10 min) in 50 mM Tris/HCl, pH 8, containing a given metal chloride (0·01–10·0 mM) before adding 5 mM substrate.

Temperature dependence of the enzyme was determined by incubating LAP (15 µg ml–1, 15–85 °C, 3 min) in 50 mM Tris/HCl and 0·5 mM MnCl2, pH 8, before addition of 5 mM L-Leu-p-NA.

Inhibition of LAP activity by peptidase inhibitors was determined by preincubation of the enzyme (15 µg ml–1, 37 °C) with EDTA, PMSF and Bestatin (Sigma) in 50 mM Tris/HCl and 0·5 mM MnCl2, pH 8, for 10 min before addition of the substrate. In the presence of metal ion chelator, MnCl2 was omitted in the assay buffer. The IC50 (the concentration of inhibitor that gave 50 % maximum inhibition) of the non-tight-binding reversible competitive inhibitor, Bestatin, was estimated from the inhibition curve.

Inhibition of H. pylori growth by Bestatin.
H. pylori (106 cells) was grown in Brucella broth medium containing 5 % FBS with or without different concentrations of Bestatin under microaerophilic conditions at 37 °C for 24 h. The growth of H. pylori was determined by measuring OD600 of the growing bacterial suspension using the UV2001 spectrophotometer.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cloning, sequencing and analysis of the H. pylori LAP gene
The LAP gene isolated from H. pylori (ATCC 43504) consists of an ORF of 1491 base pairs that encodes a polypeptide of 496 amino acids with predicted molecular mass of 54 398·61 Da. According to the nucleotide sequence retrieved from the GenBank database, this gene encodes a polypeptide chain containing an M17 LAP family signature sequence (Rawlings & Barrett, 1995). Sequence alignment analysis with other members of the M17 family suggests the existence of multiple conserved amino acid residues essential to the predicted catalytic activity (Fig. 1a). Phylogenetic comparison of the full-length H. pylori LAP sequence with six other M17 LAPs (including homologues from prokaryotic and eukaryotic organisms) indicates that the former is the least evolutionarily divergent member of this family (Fig. 1b). In addition, this enzyme exhibits low identity to other M17 LAP family members: the homology between E. coli and Haemophilus influenzae is 56 % and between E. coli and humans 30 %, while H. pylori LAP only possesses 26, 28 and 24 % identity to its counterparts from E. coli, Hae. influenzae and humans, respectively.



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Fig. 1. Relationship of H. pylori leucyl aminopeptidase (LAP) to other members of the M17 LAP family. (a) Amino acid sequence alignment of catalytic domains of the M17 cytosolic LAPs. The residues essential to metal binding (M) and catalytic activity (A) are boxed. Sequences were obtained from the GenBank database under the following accession numbers: U50151 (Lycopersicon esculentum), S65367 (Bos taurus), AF061738 (Homo sapiens), AE000496 (Escherichia coli), U32843 (Haemophilus influenzae) and AJ235270 (Rickettsia prowazekii). (b) Phylogenetic relationship of the M17 LAP family members. An unrooted dendrogram was prepared by comparing the full-length amino acid sequences of seven members of the family using CLUSTAL W alignment software from the MEGALIGN program (DNASTAR). The scale below (b) shows the proportion of substitution events as a percentage.

 
Enzymic properties of recombinant H. pylori LAP
The full-length LAP gene was expressed in E. coli as catalytically active, polyhistidine-tagged recombinant enzyme with a yield of 3–6 mg (l bacterial culture)–1. The observed molecular mass of the recombinant LAP (~55 kDa) is consistent with that calculated from the sequence of the affinity-tagged translational product (55 220·96 Da).

The LAP activity eluted in a single, well resolved peak from a Sephadex G-200 column at a molecular mass corresponding to 340 kDa. No activity was eluted at an elution volume corresponding to 55 kDa (the molecular mass of the translation products of H. pylori lap genes). These data may indicate that the translation product associates into catalytically competent homohexamers and suggests that LAP monomers may not be catalytically active. A similar observation has been reported for Leishmania Laps (Morty & Morehead, 2002).

Kinetic studies against L-Leu-p-NA suggest that H. pylori LAP is an allosteric enzyme because the V versus S plots of both uninhibited and Bestatin-inhibited LAP were not hyperbolic but sigmoid (Fig. 2a) (Dixon & Webb, 1979) with an estimated Vmax of 232 µmol min–1 (mg protein)–1. The extinction coefficient for the product p-nitroanilide is 10 µmol (A405)–1. Hill plot analysis (Fig. 2b) gave an nH value of 2·268 and an S0·5 of 0·65 mM.



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Fig. 2. Kinetics of recombinant H. pylori LAP. (a) Substrate-saturation curves of uninhibited LAP ({blacksquare}) and LAP inhibited by 50 nM Bestatin ({blacktriangleup}). Experiments were performed in 50 mM Tris/HCl (pH 8·0) with 0·5 mM MnCl2. The extinction coefficient for the product p-nitroanilide is 10 µmol A405–1. (b) Hill plot of H. pylori LAP, from which the Hill number (nH) and half-saturation constant (S0·5) were calculated. Data shown are means±SD (n=3).

 
Amidolytic activity against L-Leu-p-NA was optimal at pH 8·0 and still detectable at pH values up to 10 (Fig. 3a). It rapidly declined under moderate acidic conditions (pH 5·0). Activity could be influenced by temperature: it was optimal at 45 °C (Fig. 3b), retaining approximately 60 % activity at 100 °C. The enzyme did not precipitate under any of the experimental conditions used in this study.



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Fig. 3. Catalytic properties of recombinant H. pylori LAP. (a) Effects of pH on LAP activity against L-Leu-p-NA. The experiments were performed in AMT buffer (I=1) over the pH range between 4 and 11. (b) Effects of temperature on LAP activity against L-Leu-p-NA. The experiments were carried out in Tris/HCl buffer (pH 8) and data shown are means±SD (n=3).

 
The LAP exhibited enhanced activity in the presence of several metal ions with a rank order of manganese>cobalt>nickel (Table 1). Calcium and zinc showed inhibitory effects at 0·1 mM and abolished enzymic activity at 10 mM. After incubation with the metal-ion chelator EDTA, metal-depleted LAP (apo-LAP) displayed a negligible activity (<3 %), whereas PMSF did not demonstrate any effects.


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Table 1. Effects of divalent cations and various reagents on H. pylori LAP activity

Data shown are mean±SD (n=3).

 
Bestatin is a potent LAP inhibitor, which blocks enzymic action through a slow binding inhibition mechanism (Morty & Morehead, 2002). The IC50 of Bestatin on H. pylori LAP was 49·9 nM (Fig. 4), comparable to that estimated from the substrate-saturation curve (Fig. 2a).



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Fig. 4. Inhibition of H. pylori LAP activity by Bestatin. LAP (15 µg ml–1) was preincubated with different concentrations of Bestatin in 50 mM Tris/HCl (37 °C, pH 8·0) prior to determination of enzymic activities. Data shown are means±SD (n=3).

 
Inhibition of H. pylori growth by Bestatin
In the presence of Bestatin, the growth of H. pylori was markedly suppressed and this effect was concentration dependent (Fig. 5).



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Fig. 5. Inhibitory effects of Bestatin on the growth of H. pylori. The bacterium (106 cells) was grown in Brucella broth containing 5 % FBS, with or without different concentrations of Bestatin, under microaerophilic conditions at 37 °C for 24 h. Growth was measured by OD600 using the UV2001 spectrophotometer. Data shown are means±SD (n=3).

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
We found previously that one anti-H. pylori agent, NE-2001 (Cheng et al., 2003), was able to upregulate the expression of the LAP gene in H. pylori (data not shown). This led us to speculate that LAP may play an important role in the life cycle of this bacterium. To answer this question, we performed a series of experiments relative to the cloning, genetic analysis and biochemical characterization of LAP from a standard strain of H. pylori (ATCC 43504) as presented in this paper. To our knowledge, this is the first peptidase identified in H. pylori.

The protein encoded by the H. pylori LAP gene only possesses 20–30 % identity with other members of the M17 family of metallopeptidases. It belongs to an evolutionarily distant group of the M17 LAPs, clustering in its own branch of the phylogenetic tree (together with the plant Lycopersicon esculentum) that diverges from E. coli and Hae. influenzae. The biological significance of this genetic trait remains to be elucidated.

Optimal amidolytic activity of H. pylori LAP was observed against L-Leu-p-NA and, like other M17 LAPs, it exhibited a broad substrate specificity including Met, Arg, Ala, Ile, Val, Phe, Gly and Tyr (data not shown). This is consistent with the relatively simple genome and proteome of H. pylori. Since LAP is the only aminopeptidase in this bacterium, its broad substrate specificity ensures adequate release of various amino acids from polypeptides to maintain the life cycle.

Intriguingly, the data suggest that the kinetics of H. pylori LAP could be allosteric, which is different from that of most bacterial aminopeptidases, which show Michaelis saturation kinetics (Thierry & Janine, 1996). The only aminopeptidase that has been shown to observe allosteric kinetics is aminopeptidase A from Lactococcus lactis ssp. lactis (Niven, 1991). Like this metalloaminopeptidase, H. pylori LAP, with an nH of 2·27, is a typical allosteric enzyme in that it relies on positive co-operativity to enhance binding of the substrate. This feature implies that, due to the higher efficiency of allosteric enzymes, H. pylori LAP may play a more important role in the life cycle than LAPs in other organisms.

Metalloaminopeptidases exhibit a broad range of metal-ion dependence. M17 LAPs mainly utilize Zn(II) (Carpenter & Vahl, 1973), whereas other aminopeptidases are dependent upon Mn(II) (Cottrell et al., 2000), Fe(II) (D'Souza & Holz, 1999) and Zn(II) (Walker & Bradshaw, 1998). We have demonstrated in this study that H. pylori LAP was inactivated by incubation with metal-ion chelators and that its activity was enhanced by Mn(II), Mg(II), Co(II) and Ni(II) at millimolar concentrations. Analysis of the metal content of H. pylori LAP by ICP-AES suggests that Zn is most likely the metal cofactor for this enzyme, consistent with the observations on the bovine lens LAP (Carpenter & Vahl, 1973). Two zinc-binding sites of LAP from bovine lens (Kim & Lipscomb, 1993) and from tomato (Gu & Walling, 2002) have been identified. Site 1 readily exchanges Zn(II) for other divalent metal cations including Mn(II), Mg(II) and Co(II). Site 2 binds to Zn(II) more strongly and retains it under conditions that allow exchange of Zn(II) from site 1. It is therefore possible that activation of H. pylori LAP with Mn(II), Mg(II), Co(II) and Ni(II) might result from substitution of the site 1 Zn(II) with these ions. Substitution of the site 1 Zn(II) with Mn(II), Mg(II) and Co(II) has been shown to activate porcine kidney LAP via elevating the kcat (Van Wart & Lin, 1981). Unlike the metal chelators, PMSF did not suppress the activity of this enzyme, suggesting that no serine is involved in catalysis.

Bestatin, a potent competitive inhibitor of aminopeptidase (Wilkes & Prescott, 1985; Taylor et al., 1993), inhibited not only H. pylori LAP activity but also the growth of the bacterium. Because LAP is the only aminopeptidase in H. pylori, the bacterial growth-arresting effect of Bestatin is likely mediated through specific inhibition of LAP activity. The results point to the potential use of LAP as a drug target to develop novel anti-H. pylori agents, and Bestatin may provide a good lead for this purpose as it is well tolerated in vivo (Sakakibara et al., 1983).


   ACKNOWLEDGEMENTS
 
This work was supported by a National Natural Science Foundation of China Grant (30271555 to D. X. Z.) and a grant from the Shanghai Municipality Science & Technology Development Fund (014319238 to M. W. W.).


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Alm, R. A., Ling, L. S., Moir, D. T. & 20 other authors (1999). Genomic-sequence comparison of two unrelated isolates of the human gastric pathogen Helicobacter pylori. Nature 397, 176–180.[CrossRef][Medline]

Björkholm, B., Sjölund, M., Falk, P. G., Berg, O. G., Engstrand, L. & Andersson, D. I. (2001). Mutation frequency and biological cost of antibiotic resistance in Helicobacter pylori. Proc Natl Acad Sci U S A 98, 14607–14612.[Abstract/Free Full Text]

Booth, M., Jennings, V., Fhaolain, I. N. & O'Cuinn, G. (1990). Prolidase activity of Lactococcus lactis subsp. cremoris AM2: partial purification and characterization. J Dairy Res 57, 245–254.

Carpenter, F. H. & Vahl, J. M. (1973). Leucine aminopeptidase (bovine lens). Mechanism of activation by Mg2+ and Mn2+ of the zinc metalloenzyme, amino acid composition, and sulfhydryl content. J Biol Chem 248, 294–304.[Abstract/Free Full Text]

Cheng, N., Xie, J. S., Zhang, M. Y., Shu, C. & Zhu, D. X. (2003). A specific anti-Helicobacter pylori agent NE2001: synthesis and its effect on the growth of H. pylori. Bioorg Med Chem Lett 13, 2703–2707.[CrossRef][Medline]

Cottrell, G. S., Hooper, N. M. & Turner, A. J. (2000). Cloning, expression, and characterization of human cytosolic aminopeptidase P: a single manganese (II)-dependent enzyme. Biochemistry 39, 15121–15128.[CrossRef][Medline]

Cover, T. L. & Blaser, M. J. (1996). Helicobacter pylori infection, a paradigm for chronic mucosal inflammation: pathogenesis and implications for eradication and prevention. Adv Int Med 41, 85–117.

Dixon, M. & Webb, E. C. (1979). Enzymes, 3rd edn. London: Longman.

D'Souza, V. M. & Holz, R. C. (1999). The methionyl aminopeptidase from Escherichia coli can function as an iron (II) enzyme. Biochemistry 38, 11079–11085.[CrossRef][Medline]

Ellis, K. J. & Morrison, J. F. (1982). Buffers of constant ionic strength for studying pH-dependent processes. Methods Enzymol 87, 405–426.[Medline]

Goldberg, A. L. & Dice, J. F. (1974). Intracellular protein degradation in mammalian and bacterial cells. Annu Rev Biochem 43, 835–869.[CrossRef][Medline]

Goldberg, A. L. & John, A. C. S. (1976). Intracellular protein degradation in mammalian and bacterial cells: part 2. Annu Rev Biochem 45, 747–803.[CrossRef][Medline]

Gu, Y. G. & Walling, L. L. (2002). Identification of residues critical for activity of the wound-induced leucine aminopeptidase (LAP-A) of tomato. Eur J Biochem 269, 1630–1640.[Abstract/Free Full Text]

Hopkins, R. J., Girardi, L. S. & Turney, E. A. (1996). Relationship between Helicobacter pylori eradication and reduced duodenal and gastic ulcer recurrence: a review. Gastroenterology 110, 1244–1252.[Medline]

Howarth, J. & Lloyd, D. G. (2000). Simple 1,2-aminoalcohols as strain-specific antimalarial agents. J Antimicrob Chemother 46, 625–627.[Abstract/Free Full Text]

Kim, H. & Lipscomb, W. N. (1993). Differentiation and identification of the two catalytic metal binding sites in bovine lens leucine aminopeptidase by X-ray crystallography. Proc Natl Acad Sci U S A 90, 5006–5010.[Abstract/Free Full Text]

Knowles, G. (1993). The effects of arphamenine-A, an inhibitor of aminopeptidases, on in-vitro growth of Trypanosoma brucei brucei. J Antimicrob Chemother 32, 172–174.[Medline]

Kreiss, C., Blum, A. L. & Malfertheiner, P. (1995). Peptic ulcer pathogenesis. Curr Opin Gastroenterol 11, 25–31.

Lazdunski, A. (1989). Peptidase and protease of Escherichia coli and Salmonella typhimurium. FEMS Microbiol Rev 63, 265–276.[CrossRef]

Malfertheiner, P., Megraud, F., O'Morain, C., Hungin, A. P., Jones, R., Axon, A., Graham, D. Y. & Tytgat, G. (2002). Current concepts in the management of Helicobacter pylori infection – the Maastricht 2-2000 Consensus Report. Aliment Pharmacol Ther 16, 167–180.[Medline]

Miller, C. G. (1975). Peptidases and proteases of Escherichia coli and Salmonella typhimurium. Annu Rev Microbiol 29, 485–504.[CrossRef][Medline]

Morty, R. E. & Morehead, J. (2002). Cloning and characterization of a leucyl aminopeptidase from three pathogenic Leishmania species. J Biol Chem 277, 26057–26065.[Abstract/Free Full Text]

Müller, K. D., von Recklinghausen, G., Heintschel von Heinegg, E. & Ansorg, R. (1991). Flocculation of venereal disease research laboratory reagent by Helicobacter pylori. Eur J Microbiol Infect Dis 10, 768–770.

Nankya-Kitaka, M. F., Curley, G. P., Gavigan, C. S., Bell, A. & Dalton, J. P. (1998). Plasmodium chabaudi chabaudi and P. falciparum: inhibition of aminopeptidase and parasite growth by bestatin and nitrobestatin. Parasitol Res 84, 552–558.[CrossRef][Medline]

Niven, G. W. (1991). Purification and characterization of aminopeptidase A from Lactococcus lactis subsp. lactis NCDO712. J Gen Microbiol 137, 1207–1212.

Rawlings, N. D. & Barrett, A. J. (1995). Evolutionary families of metallopeptidases. Methods Enzymol 248, 183–228.[Medline]

Rogers, A. H., Gunadi, A., Gully, N. J. & Zilm, P. S. (1998). An aminopeptidase nutritionally important to Fusobacterium nucleatum. Microbiology 144, 1807–1813.[Medline]

Sakakibara, T., Ito, K., Irie, Y., Hagiwara, T., Sakai, Y., Hayashi, M., Kishi, H., Sakamoto, M. & Suzuki, M. (1983). Toxicological studies on bestatin. I. Acute toxicity test in mice, rats and dogs. Jpn J Antibiot 36, 2971–2984.[Medline]

Smid, E. J., Poolman, B. & Konings, W. N. (1991). Casein utilization by lactococci. Appl Environ Microbiol 57, 2447–2453.[Medline]

Tan, P. S. T. & Konings, W. N. (1990). Purification and characterization of an aminopeptidase from Lactococcus lactis subsp. cremoris Wg2. Appl Environ Microbiol 56, 526–532.

Taylor, A. (1993). Aminopeptidases: structure and function. FASEB J 7, 290–298.[Abstract/Free Full Text]

Taylor, A., Peltier, C. Z., Torre, F. J. & Hakamian, N. (1993). Inhibition of bovine lens leucine aminopeptidases by bestatin: number of binding sites and slow binding of this inhibitor. Biochemistry 32, 784–790.[CrossRef][Medline]

Thierry, G. & Janine, R. B. (1996). Bacterial aminopeptidases: properties and functions. FEMS Microbiol Rev 18, 319–344.[CrossRef][Medline]

Tomb, J. F., White, O., Kerlavage, A. R. & 39 other authors (1997). The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature 388, 539–547.[CrossRef][Medline]

Van Wart, H. E. & Lin, S. H. (1981). Metal binding stoichiometry and mechanism of metal ion modulation of the activity of porcine kidney leucine aminopeptidase. Biochemistry 20, 5682–5689.[CrossRef][Medline]

Walker, K. W. & Bradshaw, R. A. (1998). Yeast methionine aminopeptidase I can utilize either Zn2+ or Co2+ as a cofactor: a case of mistaken identity? Protein Sci 7, 2684–2687.[Abstract/Free Full Text]

Wilkes, S. H. & Prescott, J. M. (1985). The slow, tight binding of bestatin and amastatin to aminopeptidases. J Biol Chem 260, 13154–13162.[Abstract/Free Full Text]

Received 15 November 2004; revised 31 January 2005; accepted 11 February 2005.



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