Cloning, expression and characterization of insulin-degrading enzyme from tomato (Solanum lycopersicum).
A cDNA encoding insulin-degrading enzyme (IDE) was cloned from tomato (Solanum lycopersicum) and expressed in Escherichia coli in N-terminal fusion with glutathione S-transferase. GST-SlIDE was characterized as a neutral thiol-dependent metallopeptidase with insulinase activity: the recombinant enzyme cleaved the oxidized insulin B chain at eight peptide bonds, six of which are also targets of human IDE. Despite a certain preference for proline in the vicinity of the cleavage site, synthetic peptides were cleaved at apparently stochastic positions indicating that SlIDE, similar to IDEs from other organisms, does not recognize any particular amino acid motif in the primary structure of its substrates. Under steady-state conditions, an apparent K(m) of 62+/-7 microm and a catalytic efficiency (k(cat)/K(m)) of 62+/-15 mm(-1) s(-1) were determined for Abz-SKRDPPKMQTDLY(NO(3))-NH(2) as the substrate. GST-SlIDE was effectively inhibited by ATP at physiological concentrations, suggesting regulation of its activity in response to the energy status of the cell. While mammalian and plant IDEs share many of their biochemical properties, this similarity does not extend to their function in vivo, because insulin and the beta-amyloid peptide, well-established substrates of mammalian IDEs, as well as insulin-related signaling appear to be absent from plant systems.
Huet
Yoann
Y
Institute of Plant Physiology and Biotechnology, University of Hohenheim, D-70593 Stuttgart, Germany.
Strassner
Jochen
J
Schaller
Andreas
A
D000595:Amino Acid Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D007339:Insulysin / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D018551:Solanum lycopersicum / Q000235:genetics*; D008969:Molecular Sequence Data; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
D018076:DNA, Complementary; D018744:DNA, Plant; D007339:Insulysin