Portal de Periódicos da CAPES

Cloning, Expression, and Characterization of Tomato (Lycopersicon esculentum) Aminopeptidase P

2001; Elsevier BV; Volume: 276; Issue: 34 Linguagem: Inglês

10.1074/jbc.m103179200

1083-351X

Felix Hauser, Jochen Strassner, Andreas Schaller,

Ferrocene Chemistry and Applications

A cDNA (LeAPP2) was cloned from tomato coding for a 654 amino acid protein of 72.7 kDa. The deduced amino acid sequence was >40% identical with that of mammalian aminopeptidase P, a metalloexopeptidase. All amino acids reported to be important for binding of the active site metals and catalytic activity, respectively, were conserved between LeAPP2 and its mammalian homologues. LeAPP2 was expressed inEscherichia coli in N-terminal fusion with glutathioneS-transferase and was purified from bacterial extracts.LeAPP2 was verified as an aminopeptidase P, hydrolyzing the amino-terminal Xaa-Pro bonds of bradykinin and substance P. LeAPP2 also exhibited endoproteolytic activity cleaving, albeit at a reduced rate, the internal -Phe-Gly bond of substance P. Apparent K m (15.2 ± 2.4 µm) andK m/k cat (0.94 ± 0.11 mm−1 × s−1) values were obtained for H-Lys(Abz)-Pro-Pro-pNA as the substrate.LeAPP2 activity was maximally stimulated by addition of 4 mm MnCl2 and to some extent also by Mg2+, Ca2+, and Co2+, whereas other divalent metal ions (Cu2+, Zn2+) were inhibitory. Chelating agents and thiol-modifying reagents inhibited the enzyme. The data are consistent with LeAPP2 being a Mn(II)-dependent metalloprotease. This is the first characterization of a plant aminopeptidase P. A cDNA (LeAPP2) was cloned from tomato coding for a 654 amino acid protein of 72.7 kDa. The deduced amino acid sequence was >40% identical with that of mammalian aminopeptidase P, a metalloexopeptidase. All amino acids reported to be important for binding of the active site metals and catalytic activity, respectively, were conserved between LeAPP2 and its mammalian homologues. LeAPP2 was expressed inEscherichia coli in N-terminal fusion with glutathioneS-transferase and was purified from bacterial extracts.LeAPP2 was verified as an aminopeptidase P, hydrolyzing the amino-terminal Xaa-Pro bonds of bradykinin and substance P. LeAPP2 also exhibited endoproteolytic activity cleaving, albeit at a reduced rate, the internal -Phe-Gly bond of substance P. Apparent K m (15.2 ± 2.4 µm) andK m/k cat (0.94 ± 0.11 mm−1 × s−1) values were obtained for H-Lys(Abz)-Pro-Pro-pNA as the substrate.LeAPP2 activity was maximally stimulated by addition of 4 mm MnCl2 and to some extent also by Mg2+, Ca2+, and Co2+, whereas other divalent metal ions (Cu2+, Zn2+) were inhibitory. Chelating agents and thiol-modifying reagents inhibited the enzyme. The data are consistent with LeAPP2 being a Mn(II)-dependent metalloprotease. This is the first characterization of a plant aminopeptidase P. aminopeptidase P glutathione S-transferase isopropyl-1-thio-β-d-galactopyranoside matrix-assisted laser desorption ionization-time of flight/mass spectrometry polymerase chain reaction rapid amplification of cDNA ends Proline is unique among the proteinogenic amino acids in that its side chain is bonded to both the α-carbon and the amino group. The resulting cyclic structure imposes conformational restraints on proline-containing peptides relevant for structure and function of many physiologically important biomolecules. A key role for proline residues is the protection against nonspecific proteolytic degradation. Hence proline is frequently found and conserved in peptide hormones, neuropeptides, and growth factors (1Mentlein R. FEBS Lett. 1988; 2: 251-256Crossref Scopus (192) Google Scholar, 2Yaron A. Naider F. Crit. Rev. Biochem. Mol. Biol. 1993; 28: 31-81Crossref PubMed Scopus (516) Google Scholar, 3Vanhoof G. Goossens F. De Meester I. Hendriks D. Scharpé S. FASEB J. 1995; 9: 736-744Crossref PubMed Scopus (380) Google Scholar). Many bioactive polypeptides share a Xaa-Pro motif at their N termini shielding them against nonspecific N-terminal degradation. The degradation of these peptides requires proteases with specificity for the Xaa-Pro motif including proline-selective dipeptidases (dipeptidyl peptidases II and IV, cleaving the post-Pro bond) and aminopeptidase P (Xaa-Pro aminopeptidase, cleaving the pre-Pro bond). Cleavage of the Xaa-Pro motif by either one of these peptidases may initiate the proteolytic degradation/inactivation of the peptide or may result in an altered bioactivity (2Yaron A. Naider F. Crit. Rev. Biochem. Mol. Biol. 1993; 28: 31-81Crossref PubMed Scopus (516) Google Scholar, 3Vanhoof G. Goossens F. De Meester I. Hendriks D. Scharpé S. FASEB J. 1995; 9: 736-744Crossref PubMed Scopus (380) Google Scholar, 4Cunningham D.F. O'Connor B. Biochim. Biophys. Acta. 1997; 1343: 160-186Crossref PubMed Scopus (310) Google Scholar).Aminopeptidase P (APP,1 EC3.4.11.9) was first isolated from Escherichia coli (5Yaron A. Mlynar D. Biochem. Biophys. Res. Commun. 1968; 32: 658-663Crossref PubMed Scopus (120) Google Scholar) and has subsequently been characterized from many microbial and mammalian sources (reviewed in Ref. 4Cunningham D.F. O'Connor B. Biochim. Biophys. Acta. 1997; 1343: 160-186Crossref PubMed Scopus (310) Google Scholar). Mammalian APPs are now known to comprise at least two distinct forms, a cytosolic form and a membrane-bound form attached to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor (6Hooper N.M. Hryszko J. Turner A.J. Biochem. J. 1990; 267: 509-515Crossref PubMed Scopus (86) Google Scholar, 7Harbeck H.T. Mentlein R. Eur. J. Biochem. 1991; 198: 451-458Crossref PubMed Scopus (91) Google Scholar, 8Rusu I. Yaron A. Eur. J. Biochem. 1992; 210: 93-100Crossref PubMed Scopus (51) Google Scholar, 9Ryan J.W. Valido F. Berryer P. Chung A.Y.K. Ripka J.E. Biochim. Biophys. Acta. 1992; 1119: 140-147Crossref PubMed Scopus (28) Google Scholar, 10Orawski A.T. Simmons W.H. Biochemistry. 1995; 34: 11227-11236Crossref PubMed Scopus (48) Google Scholar, 11Hyde R. Hooper N.M. Turner A.J. Biochem. J. 1996; 319: 197-201Crossref PubMed Scopus (23) Google Scholar, 12Venema R.C. Ju H. Zou R. Venema V.J. Ryan J.W. Biochim. Biophys. Acta. 1997; 1354: 45-48Crossref PubMed Scopus (35) Google Scholar, 13Czirják G. Burkhart W.A. Moyer M.B. Antal J. Shears S.B. Enyedi P. Biochim. Biophys. Acta. 1999; 1444: 326-336Crossref PubMed Scopus (14) Google Scholar, 14Cottrell G.S. Hooper N.M. Turner A.J. Biochemistry. 2000; 39: 15121-15128Crossref PubMed Scopus (82) Google Scholar). APPs hydrolyze the peptide bond between any amino acid and a penultimate proline residue at the N termini of oligopeptide and protein substrates. A free amino group is required at the N terminus and the scissile bond must be in the trans configuration (15Yoshimoto T. Orawski A.T. Simmons W.H. Arch. Biochem. Biophys. 1994; 311: 28-34Crossref PubMed Scopus (63) Google Scholar). The hydrolysis of dipeptides is very slow compared with the hydrolysis of longer chains, indicating the existence of a third subsite for substrate binding, which was confirmed for E. coli and mammalian APPs (10Orawski A.T. Simmons W.H. Biochemistry. 1995; 34: 11227-11236Crossref PubMed Scopus (48) Google Scholar, 15Yoshimoto T. Orawski A.T. Simmons W.H. Arch. Biochem. Biophys. 1994; 311: 28-34Crossref PubMed Scopus (63) Google Scholar). Likely physiological substrates of APP include bradykinin, substance P, and peptide-YY (15Yoshimoto T. Orawski A.T. Simmons W.H. Arch. Biochem. Biophys. 1994; 311: 28-34Crossref PubMed Scopus (63) Google Scholar, 16Orawski A.T. Susz J.P. Simmons W.H. Mol. Cell. Biochem. 1987; 75: 123-132Crossref PubMed Scopus (50) Google Scholar, 17Medeiros M.S. Turner A.J. Biochimie (Paris). 1994; 76: 283-287Crossref PubMed Scopus (41) Google Scholar, 18Kim K.S. Kumar S. Simmons W.H. Brown N.J. J. Pharmacol. Exp. Ther. 2000; 292: 295-298PubMed Google Scholar), and APP has been implicated in the regulation of cardiovascular and pulmonary functions in vivo (19Simmons W.H. Orawski A.T. J. Biol. Chem. 1992; 267: 4897-4903Abstract Full Text PDF PubMed Google Scholar, 20Ryan J.W. Berryer P. Chung A.Y. Sheffy D.H. J. Pharmacol. Exp. Ther. 1994; 269: 941-947PubMed Google Scholar, 21Blais C. Marceau C. Rouleau J.L. Adam A. Peptides. 2000; 21: 1903-1940Crossref PubMed Scopus (119) Google Scholar).In higher plants, only very few peptides with hormone-like functions are presently known (22Schaller A. Plant Mol. Biol. 1999; 40: 763-769Crossref PubMed Scopus (24) Google Scholar, 23Ryan C.A. Pearce G. Plant Physiol. 2001; 125: 65-68Crossref PubMed Scopus (43) Google Scholar), but a more general role for peptides as signal molecules in the regulation of plant defense, growth, and development is anticipated (24Schaller A. Atta-Ur-Rahman Bioactive Natural Products. Elsevier, Amsterdam2001Google Scholar). Likewise, the proteases involved in the maturation and degradation of plant peptide hormones are still elusive. In the present work, we used a partial cDNA as a probe to isolate the cDNAs of two APPs from tomato. One of the enzymes (LeAPP2) was functionally expressed in E. coli, purified from bacterial extracts, and characterized biochemically. This is the first characterization of an APP from any plant source.DISCUSSIONAPP, like X-Pro dipeptidase (prolidase) and methionyl aminopeptidases of types I and II, belong to the M24 family in the clan MG of metalloproteases (36Rawlings N.D. Barrett A.J. Methods Enzymol. 1995; 248: 183-210Crossref PubMed Scopus (687) Google Scholar, 37Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. Academic Press, San Diego, CA1998: 1394-1411Google Scholar). Whereas the overall sequence similarity between these enzymes is rather low, their C-terminal catalytic domains share a common structural feature called the pita-bread-fold (38Bazan J.F. Weaver L.H. Roderick S.L. Huber R. Matthews B.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2473-2477Crossref PubMed Scopus (150) Google Scholar). The structures of E. coli methionyl aminopeptidase and APP have been solved and two metal ions were found to be "sandwiched" in the pita-bread domain. The metal ions are liganded by two Asp, one His, and two Glu residues, respectively, which are strictly conserved in this family of proteases (35Wilce M.C.J. Bond C.S. Dixon N.E. Freeman H.C. Guss J.M. Lilley P.E. Wilce J.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3472-3477Crossref PubMed Scopus (167) Google Scholar, 39Roderick S.L. Matthews B.W. Biochemistry. 1993; 32: 3907-3912Crossref PubMed Scopus (314) Google Scholar, 40Sprinkle T.J. Caldwell C. Ryan J.W. Arch. Biochem. Biophys. 2000; 378: 51-56Crossref PubMed Scopus (21) Google Scholar). The requirement of these residues for the catalytic activity of porcine APP has been demonstrated by site-directed mutagenesis (34Cottrell G.S. Hyde R. Lim J. Parsons M.R. Hooper N.M. Turner A.J. Biochemistry. 2000; 39: 15129-15135Crossref PubMed Scopus (21) Google Scholar). We report here the cloning and characterization of a related enzyme from tomato calledLeAPP2. This is the first characterization of an aminopeptidase P from any plant. LeAPP2 shares considerable sequence similarity with both E. coli and mammalian APPs in both the C-terminal pita-bread- as well as in the N-terminal domains. All the amino acid residues involved in metal binding (34Cottrell G.S. Hyde R. Lim J. Parsons M.R. Hooper N.M. Turner A.J. Biochemistry. 2000; 39: 15129-15135Crossref PubMed Scopus (21) Google Scholar, 35Wilce M.C.J. Bond C.S. Dixon N.E. Freeman H.C. Guss J.M. Lilley P.E. Wilce J.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3472-3477Crossref PubMed Scopus (167) Google Scholar) as well as two histidine residues implicated in proton shuttling between the solvent and the dinuclear metal center (35Wilce M.C.J. Bond C.S. Dixon N.E. Freeman H.C. Guss J.M. Lilley P.E. Wilce J.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3472-3477Crossref PubMed Scopus (167) Google Scholar) are conserved inLeAPP2 (Fig. 1).In addition to the structural similarity, LeAPP2 shares functional characteristics with known APPs. LeAPP2 expressed and purified from E. coli as a GST fusion protein exhibited APP activity, releasing the N-terminal amino acid from peptides with a penultimate proline residue. It was found to process typical substrates of mammalian APPs, i.e. bradykinin and substance P. The catalytic properties as well as structural similarity indicate a closer relationship with the cytosolic as compared with the membrane-bound forms of mammalian APPs. The pH optimum of 7.5 for LeAPP2 activity (Fig. 4 B) is consistent with its localization in the cytoplasm. Similar to the cytosolic APP from rat brain (7Harbeck H.T. Mentlein R. Eur. J. Biochem. 1991; 198: 451-458Crossref PubMed Scopus (91) Google Scholar),LeAPP2 clearly preferred Arg-Pro-Pro- (bradykinin) over Arg-Pro-Lys- (substance P), which indicates an extended binding site for recognition of the P'2 residue of the substrate as it was reported for E. coli and mammalian APPs (10Orawski A.T. Simmons W.H. Biochemistry. 1995; 34: 11227-11236Crossref PubMed Scopus (48) Google Scholar, 15Yoshimoto T. Orawski A.T. Simmons W.H. Arch. Biochem. Biophys. 1994; 311: 28-34Crossref PubMed Scopus (63) Google Scholar). Like the cytosolic APPs from E. coli, Rattus norvegicus, and Homo sapiens, but unlike the membrane-bound enzymes from R. norvegicus and Bos taurus, the tomato enzyme tolerates a Lys residue in the P'2 position.LeAPP2 also hydrolyzed the N-terminal Pro-Pro- bond of processed bradykinin, albeit at a slower rate. Cleavage of the Pro-Pro- bond at the N terminus of oligopeptide substrates has also been reported for the cytosolic rat and E. coli APPs (7Harbeck H.T. Mentlein R. Eur. J. Biochem. 1991; 198: 451-458Crossref PubMed Scopus (91) Google Scholar, 41Yoshimoto T. Murayama N. Takashi H. Tone H. Tsuru D. J. Biochem. 1988; 104: 93-97Crossref PubMed Scopus (49) Google Scholar). The rat cytosolic APP functionally expressed in E. coli, however, was found to be unable to hydrolyze the N-terminal Pro-Pro- bond of a synthetic oligopeptide substrate (13Czirják G. Burkhart W.A. Moyer M.B. Antal J. Shears S.B. Enyedi P. Biochim. Biophys. Acta. 1999; 1444: 326-336Crossref PubMed Scopus (14) Google Scholar). Endopeptidase activity, i.e. the cleavage of the -Phe-Gly- bond in substance P, appears to be a unique feature of LeAPP2. The fact that protein preparations from E. coli cultures carrying the empty expression vector, as well as cultures expressing a truncated, inactive LeAPP2, were devoid of any proteolytic activity, unequivocally shows that the observed endoproteolytic activity is a property of LeAPP2 and not that of a contaminating E. coli enzyme.There are conflicting reports in the literature with respect to the metal requirement of APPs. Until recently, supported by the crystal structures of E. coli methionyl aminopeptidase and APP, which revealed the presence of dinuclear metal centers in both enzymes (35Wilce M.C.J. Bond C.S. Dixon N.E. Freeman H.C. Guss J.M. Lilley P.E. Wilce J.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3472-3477Crossref PubMed Scopus (167) Google Scholar, 39Roderick S.L. Matthews B.W. Biochemistry. 1993; 32: 3907-3912Crossref PubMed Scopus (314) Google Scholar), two manganese (Mn(II)) or zink (Zn(II)) ions per subunit were considered necessary for maximum catalytic activity in cytosolic and membrane-bound APPs, respectively (Ref. 14Cottrell G.S. Hooper N.M. Turner A.J. Biochemistry. 2000; 39: 15121-15128Crossref PubMed Scopus (82) Google Scholar, and references therein). For methionyl aminopeptidase, on the other hand, two equivalents of cobalt (Co(II)) were proposed to be required based on the reproducible observation of highest activity in vitro in the presence of Co(II). Both the nature and the amount of metal requiredin vivo have recently been questioned, however.E. coli methionyl aminopeptidase was shown to be maximally activated upon addition of only one Fe(II) ion and iron is likely to be the in vivo ligand. Whereas the first Fe(II) ion is bound with high affinity (K d = 0.3 µm), theK d of the second metal binding site was reported to be 2.5 mm, and therefore, this site is likely to be unoccupied in vivo (42D'souza V.M. Holz R.C. Biochemistry. 1999; 38: 11079-11085Crossref PubMed Scopus (141) Google Scholar, 43D'souza V.M. Bennett B. Copik A.J. Holz R.C. Biochemistry. 2000; 39: 3817-3826Crossref PubMed Scopus (99) Google Scholar). Likewise, the two metal binding sites in human cytosolic APP (hcAPP) appear to differ in affinity. Upon expression in E. coli, this enzyme was found to contain only one equivalent of Mn(II), and this was sufficient to support proteolytic activity (14Cottrell G.S. Hooper N.M. Turner A.J. Biochemistry. 2000; 39: 15121-15128Crossref PubMed Scopus (82) Google Scholar). The hydrolysis of bradykinin and substance P by hcAPP was stimulated 2.7-fold upon further addition of Mn2+, whereas Mg2+, Ca2+, Cu2+, and Zn2+ were found to be inhibitory (in order of increasing inhibition, Ref. 14Cottrell G.S. Hooper N.M. Turner A.J. Biochemistry. 2000; 39: 15121-15128Crossref PubMed Scopus (82) Google Scholar). The effects of divalent metal ions on LeAPP2 activity are essentially the same as those observed for hcAPP (Fig. 4 A) and, therefore,LeAPP2 is also likely to be a single Mn(II)-dependent enzyme.The function of the second metal ion binding site remains obscure. Roles in the regulation of proteolytic activity or in positioning the substrate by binding its N-terminal amine group have been proposed (34Cottrell G.S. Hyde R. Lim J. Parsons M.R. Hooper N.M. Turner A.J. Biochemistry. 2000; 39: 15129-15135Crossref PubMed Scopus (21) Google Scholar,43D'souza V.M. Bennett B. Copik A.J. Holz R.C. Biochemistry. 2000; 39: 3817-3826Crossref PubMed Scopus (99) Google Scholar). A competition of substrate and metal ion for the same binding site may explain the earlier observation that the inhibitory and stimulating effects of cations on APP activity can be substrate-dependent (44Lloyd G.S. Turner A.J. Biochem. Soc. Trans. 1995; 33: 60SCrossref Scopus (3) Google Scholar, 45Lloyd G.S. Hryszko J. Hooper N.M. Turner A.J. Biochem. Pharmacol. 1996; 52: 229-236Crossref PubMed Scopus (23) Google Scholar).The enzymatic properties of LeAPP2 were further characterized using H-Lys(Abz)-Pro-Pro-pNA as the substrate for which an apparent K m of 15.2 ± 2.4 µmand a catalytic efficiency (K m/k cat) of 0.94 ± 0.11 mm−1 × s−1 were derived from steady-state kinetic analyses (Fig. 4 C). These values are within the range of catalytic constants reported for other APPs (2Yaron A. Naider F. Crit. Rev. Biochem. Mol. Biol. 1993; 28: 31-81Crossref PubMed Scopus (516) Google Scholar,7Harbeck H.T. Mentlein R. Eur. J. Biochem. 1991; 198: 451-458Crossref PubMed Scopus (91) Google Scholar, 9Ryan J.W. Valido F. Berryer P. Chung A.Y.K. Ripka J.E. Biochim. Biophys. Acta. 1992; 1119: 140-147Crossref PubMed Scopus (28) Google Scholar, 10Orawski A.T. Simmons W.H. Biochemistry. 1995; 34: 11227-11236Crossref PubMed Scopus (48) Google Scholar, 14Cottrell G.S. Hooper N.M. Turner A.J. Biochemistry. 2000; 39: 15121-15128Crossref PubMed Scopus (82) Google Scholar, 15Yoshimoto T. Orawski A.T. Simmons W.H. Arch. Biochem. Biophys. 1994; 311: 28-34Crossref PubMed Scopus (63) Google Scholar, 19Simmons W.H. Orawski A.T. J. Biol. Chem. 1992; 267: 4897-4903Abstract Full Text PDF PubMed Google Scholar). Likewise, the inhibitor profile ofLeAPP2 is typical for APPs. LeAPP2 was found to be inhibited by chelating agents with 1,10-phenanthroline being much more effective than EDTA. Consistent with the essential role of histidine residues in binding of the active site metal and in catalysis, LeAPP2 was inactivated by a histidine-modifying reagent (diethylpyrocarbonate). Inhibition by 2-mercaptoethanol andN-ethylmaleimide may indicate a functionally important cysteine residue. There is, however, no cysteine residue conserved between the two tomato and human enzymes (Fig. 1). Alternatively, thiol reagents may compete with the substrate as a ligand of the active site metal. The inhibiton by both metal chelators and thiol reagents has been reported widely for other APPs (7Harbeck H.T. Mentlein R. Eur. J. Biochem. 1991; 198: 451-458Crossref PubMed Scopus (91) Google Scholar, 8Rusu I. Yaron A. Eur. J. Biochem. 1992; 210: 93-100Crossref PubMed Scopus (51) Google Scholar, 10Orawski A.T. Simmons W.H. Biochemistry. 1995; 34: 11227-11236Crossref PubMed Scopus (48) Google Scholar, 14Cottrell G.S. Hooper N.M. Turner A.J. Biochemistry. 2000; 39: 15121-15128Crossref PubMed Scopus (82) Google Scholar, 19Simmons W.H. Orawski A.T. J. Biol. Chem. 1992; 267: 4897-4903Abstract Full Text PDF PubMed Google Scholar).The function of LeAPP2 in planta remains obscure as long as the in vivo substrate(s) are elusive. They will include oligopeptides with an amino-terminal Xaa-Pro motif. Such peptides may arise during protein degradation implying a function forLeAPP2 in protein turnover. Physiological substrates may also include plant peptide hormones implying a function forLeAPP2 in the regulation of hormone stability/activity. Considering the role of mammalian APPs in the degradation of bradykinin and substance P, it is tempting to speculate on such a function forLeAPP2. However, only very few peptide hormone-like signal molecules are known in plants (22Schaller A. Plant Mol. Biol. 1999; 40: 763-769Crossref PubMed Scopus (24) Google Scholar), and none of them contains an N-terminal Xaa-Pro motif. They are therefore not likely to be substrates of LeAPPs. Yet peptides are anticipated to play a much broader role in plant signal transduction than presently appreciated (24Schaller A. Atta-Ur-Rahman Bioactive Natural Products. Elsevier, Amsterdam2001Google Scholar), and they may require APPs for the regulation of activity. Proline is unique among the proteinogenic amino acids in that its side chain is bonded to both the α-carbon and the amino group. The resulting cyclic structure imposes conformational restraints on proline-containing peptides relevant for structure and function of many physiologically important biomolecules. A key role for proline residues is the protection against nonspecific proteolytic degradation. Hence proline is frequently found and conserved in peptide hormones, neuropeptides, and growth factors (1Mentlein R. FEBS Lett. 1988; 2: 251-256Crossref Scopus (192) Google Scholar, 2Yaron A. Naider F. Crit. Rev. Biochem. Mol. Biol. 1993; 28: 31-81Crossref PubMed Scopus (516) Google Scholar, 3Vanhoof G. Goossens F. De Meester I. Hendriks D. Scharpé S. FASEB J. 1995; 9: 736-744Crossref PubMed Scopus (380) Google Scholar). Many bioactive polypeptides share a Xaa-Pro motif at their N termini shielding them against nonspecific N-terminal degradation. The degradation of these peptides requires proteases with specificity for the Xaa-Pro motif including proline-selective dipeptidases (dipeptidyl peptidases II and IV, cleaving the post-Pro bond) and aminopeptidase P (Xaa-Pro aminopeptidase, cleaving the pre-Pro bond). Cleavage of the Xaa-Pro motif by either one of these peptidases may initiate the proteolytic degradation/inactivation of the peptide or may result in an altered bioactivity (2Yaron A. Naider F. Crit. Rev. Biochem. Mol. Biol. 1993; 28: 31-81Crossref PubMed Scopus (516) Google Scholar, 3Vanhoof G. Goossens F. De Meester I. Hendriks D. Scharpé S. FASEB J. 1995; 9: 736-744Crossref PubMed Scopus (380) Google Scholar, 4Cunningham D.F. O'Connor B. Biochim. Biophys. Acta. 1997; 1343: 160-186Crossref PubMed Scopus (310) Google Scholar). Aminopeptidase P (APP,1 EC3.4.11.9) was first isolated from Escherichia coli (5Yaron A. Mlynar D. Biochem. Biophys. Res. Commun. 1968; 32: 658-663Crossref PubMed Scopus (120) Google Scholar) and has subsequently been characterized from many microbial and mammalian sources (reviewed in Ref. 4Cunningham D.F. O'Connor B. Biochim. Biophys. Acta. 1997; 1343: 160-186Crossref PubMed Scopus (310) Google Scholar). Mammalian APPs are now known to comprise at least two distinct forms, a cytosolic form and a membrane-bound form attached to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor (6Hooper N.M. Hryszko J. Turner A.J. Biochem. J. 1990; 267: 509-515Crossref PubMed Scopus (86) Google Scholar, 7Harbeck H.T. Mentlein R. Eur. J. Biochem. 1991; 198: 451-458Crossref PubMed Scopus (91) Google Scholar, 8Rusu I. Yaron A. Eur. J. Biochem. 1992; 210: 93-100Crossref PubMed Scopus (51) Google Scholar, 9Ryan J.W. Valido F. Berryer P. Chung A.Y.K. Ripka J.E. Biochim. Biophys. Acta. 1992; 1119: 140-147Crossref PubMed Scopus (28) Google Scholar, 10Orawski A.T. Simmons W.H. Biochemistry. 1995; 34: 11227-11236Crossref PubMed Scopus (48) Google Scholar, 11Hyde R. Hooper N.M. Turner A.J. Biochem. J. 1996; 319: 197-201Crossref PubMed Scopus (23) Google Scholar, 12Venema R.C. Ju H. Zou R. Venema V.J. Ryan J.W. Biochim. Biophys. Acta. 1997; 1354: 45-48Crossref PubMed Scopus (35) Google Scholar, 13Czirják G. Burkhart W.A. Moyer M.B. Antal J. Shears S.B. Enyedi P. Biochim. Biophys. Acta. 1999; 1444: 326-336Crossref PubMed Scopus (14) Google Scholar, 14Cottrell G.S. Hooper N.M. Turner A.J. Biochemistry. 2000; 39: 15121-15128Crossref PubMed Scopus (82) Google Scholar). APPs hydrolyze the peptide bond between any amino acid and a penultimate proline residue at the N termini of oligopeptide and protein substrates. A free amino group is required at the N terminus and the scissile bond must be in the trans configuration (15Yoshimoto T. Orawski A.T. Simmons W.H. Arch. Biochem. Biophys. 1994; 311: 28-34Crossref PubMed Scopus (63) Google Scholar). The hydrolysis of dipeptides is very slow compared with the hydrolysis of longer chains, indicating the existence of a third subsite for substrate binding, which was confirmed for E. coli and mammalian APPs (10Orawski A.T. Simmons W.H. Biochemistry. 1995; 34: 11227-11236Crossref PubMed Scopus (48) Google Scholar, 15Yoshimoto T. Orawski A.T. Simmons W.H. Arch. Biochem. Biophys. 1994; 311: 28-34Crossref PubMed Scopus (63) Google Scholar). Likely physiological substrates of APP include bradykinin, substance P, and peptide-YY (15Yoshimoto T. Orawski A.T. Simmons W.H. Arch. Biochem. Biophys. 1994; 311: 28-34Crossref PubMed Scopus (63) Google Scholar, 16Orawski A.T. Susz J.P. Simmons W.H. Mol. Cell. Biochem. 1987; 75: 123-132Crossref PubMed Scopus (50) Google Scholar, 17Medeiros M.S. Turner A.J. Biochimie (Paris). 1994; 76: 283-287Crossref PubMed Scopus (41) Google Scholar, 18Kim K.S. Kumar S. Simmons W.H. Brown N.J. J. Pharmacol. Exp. Ther. 2000; 292: 295-298PubMed Google Scholar), and APP has been implicated in the regulation of cardiovascular and pulmonary functions in vivo (19Simmons W.H. Orawski A.T. J. Biol. Chem. 1992; 267: 4897-4903Abstract Full Text PDF PubMed Google Scholar, 20Ryan J.W. Berryer P. Chung A.Y. Sheffy D.H. J. Pharmacol. Exp. Ther. 1994; 269: 941-947PubMed Google Scholar, 21Blais C. Marceau C. Rouleau J.L. Adam A. Peptides. 2000; 21: 1903-1940Crossref PubMed Scopus (119) Google Scholar). In higher plants, only very few peptides with hormone-like functions are presently known (22Schaller A. Plant Mol. Biol. 1999; 40: 763-769Crossref PubMed Scopus (24) Google Scholar, 23Ryan C.A. Pearce G. Plant Physiol. 2001; 125: 65-68Crossref PubMed Scopus (43) Google Scholar), but a more general role for peptides as signal molecules in the regulation of plant defense, growth, and development is anticipated (24Schaller A. Atta-Ur-Rahman Bioactive Natural Products. Elsevier, Amsterdam2001Google Scholar). Likewise, the proteases involved in the maturation and degradation of plant peptide hormones are still elusive. In the present work, we used a partial cDNA as a probe to isolate the cDNAs of two APPs from tomato. One of the enzymes (LeAPP2) was functionally expressed in E. coli, purified from bacterial extracts, and characterized biochemically. This is the first characterization of an APP from any plant source. DISCUSSIONAPP, like X-Pro dipeptidase (prolidase) and methionyl aminopeptidases of types I and II, belong to the M24 family in the clan MG of metalloproteases (36Rawlings N.D. Barrett A.J. Methods Enzymol. 1995; 248: 183-210Crossref PubMed Scopus (687) Google Scholar, 37Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. Academic Press, San Diego, CA1998: 1394-1411Google Scholar). Whereas the overall sequence similarity between these enzymes is rather low, their C-terminal catalytic domains share a common structural feature called the pita-bread-fold (38Bazan J.F. Weaver L.H. Roderick S.L. Huber R. Matthews B.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2473-2477Crossref PubMed Scopus (150) Google Scholar). The structures of E. coli methionyl aminopeptidase and APP have been solved and two metal ions were found to be "sandwiched" in the pita-bread domain. The metal ions are liganded by two Asp, one His, and two Glu residues, respectively, which are strictly conserved in this family of proteases (35Wilce M.C.J. Bond C.S. Dixon N.E. Freeman H.C. Guss J.M. Lilley P.E. Wilce J.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3472-3477Crossref PubMed Scopus (167) Google Scholar, 39Roderick S.L. Matthews B.W. Biochemistry. 1993; 32: 3907-3912Crossref PubMed Scopus (314) Google Scholar, 40Sprinkle T.J. Caldwell C. Ryan J.W. Arch. Biochem. Biophys. 2000; 378: 51-56Crossref PubMed Scopus (21) Google Scholar). The requirement of these residues for the catalytic activity of porcine APP has been demonstrated by site-directed mutagenesis (34Cottrell G.S. Hyde R. Lim J. Parsons M.R. Hooper N.M. Turner A.J. Biochemistry. 2000; 39: 15129-15135Crossref PubMed Scopus (21) Google Scholar). We report here the cloning and characterization of a related enzyme from tomato calledLeAPP2. This is the first characterization of an aminopeptidase P from any plant. LeAPP2 shares considerable sequence similarity with both E. coli and mammalian APPs in both the C-terminal pita-bread- as well as in the N-terminal domains. All the amino acid residues involved in metal binding (34Cottrell G.S. Hyde R. Lim J. Parsons M.R. Hooper N.M. Turner A.J. Biochemistry. 2000; 39: 15129-15135Crossref PubMed Scopus (21) Google Scholar, 35Wilce M.C.J. Bond C.S. Dixon N.E. Freeman H.C. Guss J.M. Lilley P.E. Wilce J.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3472-3477Crossref PubMed Scopus (167) Google Scholar) as well as two histidine residues implicated in proton shuttling between the solvent and the dinuclear metal center (35Wilce M.C.J. Bond C.S. Dixon N.E. Freeman H.C. Guss J.M. Lilley P.E. Wilce J.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3472-3477Crossref PubMed Scopus (167) Google Scholar) are conserved inLeAPP2 (Fig. 1).In addition to the structural similarity, LeAPP2 shares functional characteristics with known APPs. LeAPP2 expressed and purified from E. coli as a GST fusion protein exhibited APP activity, releasing the N-terminal amino acid from peptides with a penultimate proline residue. It was found to process typical substrates of mammalian APPs, i.e. bradykinin and substance P. The catalytic properties as well as structural similarity indicate a closer relationship with the cytosolic as compared with the membrane-bound forms of mammalian APPs. The pH optimum of 7.5 for LeAPP2 activity (Fig. 4 B) is consistent with its localization in the cytoplasm. Similar to the cytosolic APP from rat brain (7Harbeck H.T. Mentlein R. Eur. J. Biochem. 1991; 198: 451-458Crossref PubMed Scopus (91) Google Scholar),LeAPP2 clearly preferred Arg-Pro-Pro- (bradykinin) over Arg-Pro-Lys- (substance P), which indicates an extended binding site for recognition of the P'2 residue of the substrate as it was reported for E. coli and mammalian APPs (10Orawski A.T. Simmons W.H. Biochemistry. 1995; 34: 11227-11236Crossref PubMed Scopus (48) Google Scholar, 15Yoshimoto T. Orawski A.T. Simmons W.H. Arch. Biochem. Biophys. 1994; 311: 28-34Crossref PubMed Scopus (63) Google Scholar). Like the cytosolic APPs from E. coli, Rattus norvegicus, and Homo sapiens, but unlike the membrane-bound enzymes from R. norvegicus and Bos taurus, the tomato enzyme tolerates a Lys residue in the P'2 position.LeAPP2 also hydrolyzed the N-terminal Pro-Pro- bond of processed bradykinin, albeit at a slower rate. Cleavage of the Pro-Pro- bond at the N terminus of oligopeptide substrates has also been reported for the cytosolic rat and E. coli APPs (7Harbeck H.T. Mentlein R. Eur. J. Biochem. 1991; 198: 451-458Crossref PubMed Scopus (91) Google Scholar, 41Yoshimoto T. Murayama N. Takashi H. Tone H. Tsuru D. J. Biochem. 1988; 104: 93-97Crossref PubMed Scopus (49) Google Scholar). The rat cytosolic APP functionally expressed in E. coli, however, was found to be unable to hydrolyze the N-terminal Pro-Pro- bond of a synthetic oligopeptide substrate (13Czirják G. Burkhart W.A. Moyer M.B. Antal J. Shears S.B. Enyedi P. Biochim. Biophys. Acta. 1999; 1444: 326-336Crossref PubMed Scopus (14) Google Scholar). Endopeptidase activity, i.e. the cleavage of the -Phe-Gly- bond in substance P, appears to be a unique feature of LeAPP2. The fact that protein preparations from E. coli cultures carrying the empty expression vector, as well as cultures expressing a truncated, inactive LeAPP2, were devoid of any proteolytic activity, unequivocally shows that the observed endoproteolytic activity is a property of LeAPP2 and not that of a contaminating E. coli enzyme.There are conflicting reports in the literature with respect to the metal requirement of APPs. Until recently, supported by the crystal structures of E. coli methionyl aminopeptidase and APP, which revealed the presence of dinuclear metal centers in both enzymes (35Wilce M.C.J. Bond C.S. Dixon N.E. Freeman H.C. Guss J.M. Lilley P.E. Wilce J.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3472-3477Crossref PubMed Scopus (167) Google Scholar, 39Roderick S.L. Matthews B.W. Biochemistry. 1993; 32: 3907-3912Crossref PubMed Scopus (314) Google Scholar), two manganese (Mn(II)) or zink (Zn(II)) ions per subunit were considered necessary for maximum catalytic activity in cytosolic and membrane-bound APPs, respectively (Ref. 14Cottrell G.S. Hooper N.M. Turner A.J. Biochemistry. 2000; 39: 15121-15128Crossref PubMed Scopus (82) Google Scholar, and references therein). For methionyl aminopeptidase, on the other hand, two equivalents of cobalt (Co(II)) were proposed to be required based on the reproducible observation of highest activity in vitro in the presence of Co(II). Both the nature and the amount of metal requiredin vivo have recently been questioned, however.E. coli methionyl aminopeptidase was shown to be maximally activated upon addition of only one Fe(II) ion and iron is likely to be the in vivo ligand. Whereas the first Fe(II) ion is bound with high affinity (K d = 0.3 µm), theK d of the second metal binding site was reported to be 2.5 mm, and therefore, this site is likely to be unoccupied in vivo (42D'souza V.M. Holz R.C. Biochemistry. 1999; 38: 11079-11085Crossref PubMed Scopus (141) Google Scholar, 43D'souza V.M. Bennett B. Copik A.J. Holz R.C. Biochemistry. 2000; 39: 3817-3826Crossref PubMed Scopus (99) Google Scholar). Likewise, the two metal binding sites in human cytosolic APP (hcAPP) appear to differ in affinity. Upon expression in E. coli, this enzyme was found to contain only one equivalent of Mn(II), and this was sufficient to support proteolytic activity (14Cottrell G.S. Hooper N.M. Turner A.J. Biochemistry. 2000; 39: 15121-15128Crossref PubMed Scopus (82) Google Scholar). The hydrolysis of bradykinin and substance P by hcAPP was stimulated 2.7-fold upon further addition of Mn2+, whereas Mg2+, Ca2+, Cu2+, and Zn2+ were found to be inhibitory (in order of increasing inhibition, Ref. 14Cottrell G.S. Hooper N.M. Turner A.J. Biochemistry. 2000; 39: 15121-15128Crossref PubMed Scopus (82) Google Scholar). The effects of divalent metal ions on LeAPP2 activity are essentially the same as those observed for hcAPP (Fig. 4 A) and, therefore,LeAPP2 is also likely to be a single Mn(II)-dependent enzyme.The function of the second metal ion binding site remains obscure. Roles in the regulation of proteolytic activity or in positioning the substrate by binding its N-terminal amine group have been proposed (34Cottrell G.S. Hyde R. Lim J. Parsons M.R. Hooper N.M. Turner A.J. Biochemistry. 2000; 39: 15129-15135Crossref PubMed Scopus (21) Google Scholar,43D'souza V.M. Bennett B. Copik A.J. Holz R.C. Biochemistry. 2000; 39: 3817-3826Crossref PubMed Scopus (99) Google Scholar). A competition of substrate and metal ion for the same binding site may explain the earlier observation that the inhibitory and stimulating effects of cations on APP activity can be substrate-dependent (44Lloyd G.S. Turner A.J. Biochem. Soc. Trans. 1995; 33: 60SCrossref Scopus (3) Google Scholar, 45Lloyd G.S. Hryszko J. Hooper N.M. Turner A.J. Biochem. Pharmacol. 1996; 52: 229-236Crossref PubMed Scopus (23) Google Scholar).The enzymatic properties of LeAPP2 were further characterized using H-Lys(Abz)-Pro-Pro-pNA as the substrate for which an apparent K m of 15.2 ± 2.4 µmand a catalytic efficiency (K m/k cat) of 0.94 ± 0.11 mm−1 × s−1 were derived from steady-state kinetic analyses (Fig. 4 C). These values are within the range of catalytic constants reported for other APPs (2Yaron A. Naider F. Crit. Rev. Biochem. Mol. Biol. 1993; 28: 31-81Crossref PubMed Scopus (516) Google Scholar,7Harbeck H.T. Mentlein R. Eur. J. Biochem. 1991; 198: 451-458Crossref PubMed Scopus (91) Google Scholar, 9Ryan J.W. Valido F. Berryer P. Chung A.Y.K. Ripka J.E. Biochim. Biophys. Acta. 1992; 1119: 140-147Crossref PubMed Scopus (28) Google Scholar, 10Orawski A.T. Simmons W.H. Biochemistry. 1995; 34: 11227-11236Crossref PubMed Scopus (48) Google Scholar, 14Cottrell G.S. Hooper N.M. Turner A.J. Biochemistry. 2000; 39: 15121-15128Crossref PubMed Scopus (82) Google Scholar, 15Yoshimoto T. Orawski A.T. Simmons W.H. Arch. Biochem. Biophys. 1994; 311: 28-34Crossref PubMed Scopus (63) Google Scholar, 19Simmons W.H. Orawski A.T. J. Biol. Chem. 1992; 267: 4897-4903Abstract Full Text PDF PubMed Google Scholar). Likewise, the inhibitor profile ofLeAPP2 is typical for APPs. LeAPP2 was found to be inhibited by chelating agents with 1,10-phenanthroline being much more effective than EDTA. Consistent with the essential role of histidine residues in binding of the active site metal and in catalysis, LeAPP2 was inactivated by a histidine-modifying reagent (diethylpyrocarbonate). Inhibition by 2-mercaptoethanol andN-ethylmaleimide may indicate a functionally important cysteine residue. There is, however, no cysteine residue conserved between the two tomato and human enzymes (Fig. 1). Alternatively, thiol reagents may compete with the substrate as a ligand of the active site metal. The inhibiton by both metal chelators and thiol reagents has been reported widely for other APPs (7Harbeck H.T. Mentlein R. Eur. J. Biochem. 1991; 198: 451-458Crossref PubMed Scopus (91) Google Scholar, 8Rusu I. Yaron A. Eur. J. Biochem. 1992; 210: 93-100Crossref PubMed Scopus (51) Google Scholar, 10Orawski A.T. Simmons W.H. Biochemistry. 1995; 34: 11227-11236Crossref PubMed Scopus (48) Google Scholar, 14Cottrell G.S. Hooper N.M. Turner A.J. Biochemistry. 2000; 39: 15121-15128Crossref PubMed Scopus (82) Google Scholar, 19Simmons W.H. Orawski A.T. J. Biol. Chem. 1992; 267: 4897-4903Abstract Full Text PDF PubMed Google Scholar).The function of LeAPP2 in planta remains obscure as long as the in vivo substrate(s) are elusive. They will include oligopeptides with an amino-terminal Xaa-Pro motif. Such peptides may arise during protein degradation implying a function forLeAPP2 in protein turnover. Physiological substrates may also include plant peptide hormones implying a function forLeAPP2 in the regulation of hormone stability/activity. Considering the role of mammalian APPs in the degradation of bradykinin and substance P, it is tempting to speculate on such a function forLeAPP2. However, only very few peptide hormone-like signal molecules are known in plants (22Schaller A. Plant Mol. Biol. 1999; 40: 763-769Crossref PubMed Scopus (24) Google Scholar), and none of them contains an N-terminal Xaa-Pro motif. They are therefore not likely to be substrates of LeAPPs. Yet peptides are anticipated to play a much broader role in plant signal transduction than presently appreciated (24Schaller A. Atta-Ur-Rahman Bioactive Natural Products. Elsevier, Amsterdam2001Google Scholar), and they may require APPs for the regulation of activity. APP, like X-Pro dipeptidase (prolidase) and methionyl aminopeptidases of types I and II, belong to the M24 family in the clan MG of metalloproteases (36Rawlings N.D. Barrett A.J. Methods Enzymol. 1995; 248: 183-210Crossref PubMed Scopus (687) Google Scholar, 37Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. Academic Press, San Diego, CA1998: 1394-1411Google Scholar). Whereas the overall sequence similarity between these enzymes is rather low, their C-terminal catalytic domains share a common structural feature called the pita-bread-fold (38Bazan J.F. Weaver L.H. Roderick S.L. Huber R. Matthews B.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2473-2477Crossref PubMed Scopus (150) Google Scholar). The structures of E. coli methionyl aminopeptidase and APP have been solved and two metal ions were found to be "sandwiched" in the pita-bread domain. The metal ions are liganded by two Asp, one His, and two Glu residues, respectively, which are strictly conserved in this family of proteases (35Wilce M.C.J. Bond C.S. Dixon N.E. Freeman H.C. Guss J.M. Lilley P.E. Wilce J.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3472-3477Crossref PubMed Scopus (167) Google Scholar, 39Roderick S.L. Matthews B.W. Biochemistry. 1993; 32: 3907-3912Crossref PubMed Scopus (314) Google Scholar, 40Sprinkle T.J. Caldwell C. Ryan J.W. Arch. Biochem. Biophys. 2000; 378: 51-56Crossref PubMed Scopus (21) Google Scholar). The requirement of these residues for the catalytic activity of porcine APP has been demonstrated by site-directed mutagenesis (34Cottrell G.S. Hyde R. Lim J. Parsons M.R. Hooper N.M. Turner A.J. Biochemistry. 2000; 39: 15129-15135Crossref PubMed Scopus (21) Google Scholar). We report here the cloning and characterization of a related enzyme from tomato calledLeAPP2. This is the first characterization of an aminopeptidase P from any plant. LeAPP2 shares considerable sequence similarity with both E. coli and mammalian APPs in both the C-terminal pita-bread- as well as in the N-terminal domains. All the amino acid residues involved in metal binding (34Cottrell G.S. Hyde R. Lim J. Parsons M.R. Hooper N.M. Turner A.J. Biochemistry. 2000; 39: 15129-15135Crossref PubMed Scopus (21) Google Scholar, 35Wilce M.C.J. Bond C.S. Dixon N.E. Freeman H.C. Guss J.M. Lilley P.E. Wilce J.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3472-3477Crossref PubMed Scopus (167) Google Scholar) as well as two histidine residues implicated in proton shuttling between the solvent and the dinuclear metal center (35Wilce M.C.J. Bond C.S. Dixon N.E. Freeman H.C. Guss J.M. Lilley P.E. Wilce J.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3472-3477Crossref PubMed Scopus (167) Google Scholar) are conserved inLeAPP2 (Fig. 1). In addition to the structural similarity, LeAPP2 shares functional characteristics with known APPs. LeAPP2 expressed and purified from E. coli as a GST fusion protein exhibited APP activity, releasing the N-terminal amino acid from peptides with a penultimate proline residue. It was found to process typical substrates of mammalian APPs, i.e. bradykinin and substance P. The catalytic properties as well as structural similarity indicate a closer relationship with the cytosolic as compared with the membrane-bound forms of mammalian APPs. The pH optimum of 7.5 for LeAPP2 activity (Fig. 4 B) is consistent with its localization in the cytoplasm. Similar to the cytosolic APP from rat brain (7Harbeck H.T. Mentlein R. Eur. J. Biochem. 1991; 198: 451-458Crossref PubMed Scopus (91) Google Scholar),LeAPP2 clearly preferred Arg-Pro-Pro- (bradykinin) over Arg-Pro-Lys- (substance P), which indicates an extended binding site for recognition of the P'2 residue of the substrate as it was reported for E. coli and mammalian APPs (10Orawski A.T. Simmons W.H. Biochemistry. 1995; 34: 11227-11236Crossref PubMed Scopus (48) Google Scholar, 15Yoshimoto T. Orawski A.T. Simmons W.H. Arch. Biochem. Biophys. 1994; 311: 28-34Crossref PubMed Scopus (63) Google Scholar). Like the cytosolic APPs from E. coli, Rattus norvegicus, and Homo sapiens, but unlike the membrane-bound enzymes from R. norvegicus and Bos taurus, the tomato enzyme tolerates a Lys residue in the P'2 position. LeAPP2 also hydrolyzed the N-terminal Pro-Pro- bond of processed bradykinin, albeit at a slower rate. Cleavage of the Pro-Pro- bond at the N terminus of oligopeptide substrates has also been reported for the cytosolic rat and E. coli APPs (7Harbeck H.T. Mentlein R. Eur. J. Biochem. 1991; 198: 451-458Crossref PubMed Scopus (91) Google Scholar, 41Yoshimoto T. Murayama N. Takashi H. Tone H. Tsuru D. J. Biochem. 1988; 104: 93-97Crossref PubMed Scopus (49) Google Scholar). The rat cytosolic APP functionally expressed in E. coli, however, was found to be unable to hydrolyze the N-terminal Pro-Pro- bond of a synthetic oligopeptide substrate (13Czirják G. Burkhart W.A. Moyer M.B. Antal J. Shears S.B. Enyedi P. Biochim. Biophys. Acta. 1999; 1444: 326-336Crossref PubMed Scopus (14) Google Scholar). Endopeptidase activity, i.e. the cleavage of the -Phe-Gly- bond in substance P, appears to be a unique feature of LeAPP2. The fact that protein preparations from E. coli cultures carrying the empty expression vector, as well as cultures expressing a truncated, inactive LeAPP2, were devoid of any proteolytic activity, unequivocally shows that the observed endoproteolytic activity is a property of LeAPP2 and not that of a contaminating E. coli enzyme. There are conflicting reports in the literature with respect to the metal requirement of APPs. Until recently, supported by the crystal structures of E. coli methionyl aminopeptidase and APP, which revealed the presence of dinuclear metal centers in both enzymes (35Wilce M.C.J. Bond C.S. Dixon N.E. Freeman H.C. Guss J.M. Lilley P.E. Wilce J.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3472-3477Crossref PubMed Scopus (167) Google Scholar, 39Roderick S.L. Matthews B.W. Biochemistry. 1993; 32: 3907-3912Crossref PubMed Scopus (314) Google Scholar), two manganese (Mn(II)) or zink (Zn(II)) ions per subunit were considered necessary for maximum catalytic activity in cytosolic and membrane-bound APPs, respectively (Ref. 14Cottrell G.S. Hooper N.M. Turner A.J. Biochemistry. 2000; 39: 15121-15128Crossref PubMed Scopus (82) Google Scholar, and references therein). For methionyl aminopeptidase, on the other hand, two equivalents of cobalt (Co(II)) were proposed to be required based on the reproducible observation of highest activity in vitro in the presence of Co(II). Both the nature and the amount of metal requiredin vivo have recently been questioned, however. E. coli methionyl aminopeptidase was shown to be maximally activated upon addition of only one Fe(II) ion and iron is likely to be the in vivo ligand. Whereas the first Fe(II) ion is bound with high affinity (K d = 0.3 µm), theK d of the second metal binding site was reported to be 2.5 mm, and therefore, this site is likely to be unoccupied in vivo (42D'souza V.M. Holz R.C. Biochemistry. 1999; 38: 11079-11085Crossref PubMed Scopus (141) Google Scholar, 43D'souza V.M. Bennett B. Copik A.J. Holz R.C. Biochemistry. 2000; 39: 3817-3826Crossref PubMed Scopus (99) Google Scholar). Likewise, the two metal binding sites in human cytosolic APP (hcAPP) appear to differ in affinity. Upon expression in E. coli, this enzyme was found to contain only one equivalent of Mn(II), and this was sufficient to support proteolytic activity (14Cottrell G.S. Hooper N.M. Turner A.J. Biochemistry. 2000; 39: 15121-15128Crossref PubMed Scopus (82) Google Scholar). The hydrolysis of bradykinin and substance P by hcAPP was stimulated 2.7-fold upon further addition of Mn2+, whereas Mg2+, Ca2+, Cu2+, and Zn2+ were found to be inhibitory (in order of increasing inhibition, Ref. 14Cottrell G.S. Hooper N.M. Turner A.J. Biochemistry. 2000; 39: 15121-15128Crossref PubMed Scopus (82) Google Scholar). The effects of divalent metal ions on LeAPP2 activity are essentially the same as those observed for hcAPP (Fig. 4 A) and, therefore,LeAPP2 is also likely to be a single Mn(II)-dependent enzyme. The function of the second metal ion binding site remains obscure. Roles in the regulation of proteolytic activity or in positioning the substrate by binding its N-terminal amine group have been proposed (34Cottrell G.S. Hyde R. Lim J. Parsons M.R. Hooper N.M. Turner A.J. Biochemistry. 2000; 39: 15129-15135Crossref PubMed Scopus (21) Google Scholar,43D'souza V.M. Bennett B. Copik A.J. Holz R.C. Biochemistry. 2000; 39: 3817-3826Crossref PubMed Scopus (99) Google Scholar). A competition of substrate and metal ion for the same binding site may explain the earlier observation that the inhibitory and stimulating effects of cations on APP activity can be substrate-dependent (44Lloyd G.S. Turner A.J. Biochem. Soc. Trans. 1995; 33: 60SCrossref Scopus (3) Google Scholar, 45Lloyd G.S. Hryszko J. Hooper N.M. Turner A.J. Biochem. Pharmacol. 1996; 52: 229-236Crossref PubMed Scopus (23) Google Scholar). The enzymatic properties of LeAPP2 were further characterized using H-Lys(Abz)-Pro-Pro-pNA as the substrate for which an apparent K m of 15.2 ± 2.4 µmand a catalytic efficiency (K m/k cat) of 0.94 ± 0.11 mm−1 × s−1 were derived from steady-state kinetic analyses (Fig. 4 C). These values are within the range of catalytic constants reported for other APPs (2Yaron A. Naider F. Crit. Rev. Biochem. Mol. Biol. 1993; 28: 31-81Crossref PubMed Scopus (516) Google Scholar,7Harbeck H.T. Mentlein R. Eur. J. Biochem. 1991; 198: 451-458Crossref PubMed Scopus (91) Google Scholar, 9Ryan J.W. Valido F. Berryer P. Chung A.Y.K. Ripka J.E. Biochim. Biophys. Acta. 1992; 1119: 140-147Crossref PubMed Scopus (28) Google Scholar, 10Orawski A.T. Simmons W.H. Biochemistry. 1995; 34: 11227-11236Crossref PubMed Scopus (48) Google Scholar, 14Cottrell G.S. Hooper N.M. Turner A.J. Biochemistry. 2000; 39: 15121-15128Crossref PubMed Scopus (82) Google Scholar, 15Yoshimoto T. Orawski A.T. Simmons W.H. Arch. Biochem. Biophys. 1994; 311: 28-34Crossref PubMed Scopus (63) Google Scholar, 19Simmons W.H. Orawski A.T. J. Biol. Chem. 1992; 267: 4897-4903Abstract Full Text PDF PubMed Google Scholar). Likewise, the inhibitor profile ofLeAPP2 is typical for APPs. LeAPP2 was found to be inhibited by chelating agents with 1,10-phenanthroline being much more effective than EDTA. Consistent with the essential role of histidine residues in binding of the active site metal and in catalysis, LeAPP2 was inactivated by a histidine-modifying reagent (diethylpyrocarbonate). Inhibition by 2-mercaptoethanol andN-ethylmaleimide may indicate a functionally important cysteine residue. There is, however, no cysteine residue conserved between the two tomato and human enzymes (Fig. 1). Alternatively, thiol reagents may compete with the substrate as a ligand of the active site metal. The inhibiton by both metal chelators and thiol reagents has been reported widely for other APPs (7Harbeck H.T. Mentlein R. Eur. J. Biochem. 1991; 198: 451-458Crossref PubMed Scopus (91) Google Scholar, 8Rusu I. Yaron A. Eur. J. Biochem. 1992; 210: 93-100Crossref PubMed Scopus (51) Google Scholar, 10Orawski A.T. Simmons W.H. Biochemistry. 1995; 34: 11227-11236Crossref PubMed Scopus (48) Google Scholar, 14Cottrell G.S. Hooper N.M. Turner A.J. Biochemistry. 2000; 39: 15121-15128Crossref PubMed Scopus (82) Google Scholar, 19Simmons W.H. Orawski A.T. J. Biol. Chem. 1992; 267: 4897-4903Abstract Full Text PDF PubMed Google Scholar). The function of LeAPP2 in planta remains obscure as long as the in vivo substrate(s) are elusive. They will include oligopeptides with an amino-terminal Xaa-Pro motif. Such peptides may arise during protein degradation implying a function forLeAPP2 in protein turnover. Physiological substrates may also include plant peptide hormones implying a function forLeAPP2 in the regulation of hormone stability/activity. Considering the role of mammalian APPs in the degradation of bradykinin and substance P, it is tempting to speculate on such a function forLeAPP2. However, only very few peptide hormone-like signal molecules are known in plants (22Schaller A. Plant Mol. Biol. 1999; 40: 763-769Crossref PubMed Scopus (24) Google Scholar), and none of them contains an N-terminal Xaa-Pro motif. They are therefore not likely to be substrates of LeAPPs. Yet peptides are anticipated to play a much broader role in plant signal transduction than presently appreciated (24Schaller A. Atta-Ur-Rahman Bioactive Natural Products. Elsevier, Amsterdam2001Google Scholar), and they may require APPs for the regulation of activity. We thank Dr. Peter Macheroux (ETH Zürich) for help with the MALDI-TOF/MS experiments and D. Frasson for excellent technical assistance.