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

Abstract

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.