Cloning, expression, and characterization of tomato (Lycopersicon esculentum) 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 in Escherichia coli in N-terminal fusion with glutathione S-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 microm) and K(m)/k(cat) (0.94 +/- 0.11 mm(-1) x 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 MnCl(2) and to some extent also by Mg(2+), Ca(2+), and Co(2+), whereas other divalent metal ions (Cu(2+), Zn(2+)) 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.
Hauser
F
F
Institute of Plant Sciences, Swiss Federal Institute of Technology Zürich, Universitätstrasse 2, CH-8092 Zürich, Switzerland.
Strassner
J
J
Schaller
A
A
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D017343:Genes, Plant; D018551:Solanum lycopersicum / Q000201:enzymology*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
D017931:DNA Primers; D018076:DNA, Complementary; D011994:Recombinant Proteins; D000626:Aminopeptidases; C022750:X-Pro aminopeptidase