Overexpression, purification and biochemical characterization of the wound-induced leucine aminopeptidase of tomato.
Wounding of tomato leaves results in the accumulation of an exoprotease called leucine aminopeptidase (LAP-A). While the expression of LapA genes are well characterized, the specificity of the LAP-A enzyme has not been studied. The LAP-A preprotein and mature polypeptide were overexpressed in Escherichia coli. PreLAP-A was not processed and was inactive accumulating in inclusion bodies. In contrast, 55-kDa mature LAP-A subunits assembled into an active, 357-kDa enzyme in E. coli. LAP-A from E. coli cultures was purified to apparent homogeneity and characterized relative to its animal (porcine LAP) and prokaryotic (E. coli PepA) homologues. Similar to the porcine and E. coli enzymes, the tomato LAP-A had high temperature and pH optima. Mn2+ was a strong activator for all three enzymes, while chelators, zinc ion, and the slow-binding aminopeptidase inhibitors (amastatin and bestatin) strongly inhibited activities of all three LAPs. The substrate specificities of porcine, E. coli and tomato LAPs were determined using amino-acid-p-nitroanilide and -beta-naphthylamide substrates. The tomato LAP-A preferentially hydrolyzed substrates with N-terminal Leu, Met and Arg residues. LAP-A had substantially lower levels of activity on other chromogenic substrates. Several differences in substrate specificities for the animal, plant and prokaryotic enzymes were noted.
Gu
Y Q
YQ
Department of Botany, University of California, Riverside, CA, USA.
Holzer
F M
FM
Walling
L L
LL
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D003001:Cloning, Molecular; D004926:Escherichia coli; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D006358:Hot Temperature; D007700:Kinetics; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D007931:Leucyl Aminopeptidase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D018551:Solanum lycopersicum / Q000201:enzymology / Q000235:genetics / Q000502:physiology*; D046911:Macromolecular Substances; D008345:Manganese / Q000494:pharmacology; D008970:Molecular Weight; D010455:Peptides; D018515:Plant Leaves; D011480:Protease Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D013552:Swine
D000900:Anti-Bacterial Agents; D046911:Macromolecular Substances; D010455:Peptides; D011480:Protease Inhibitors; D008345:Manganese; C016977:amastatin; D007931:Leucyl Aminopeptidase; D007930:Leucine; C012211:ubenimex