Overexpression, purification and biochemical characterization of the wound‐induced leucine aminopeptidase of tomato

Abstract

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. Mn 2+ 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 -b-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.

Plant Leucine Aminopeptidases Moonlight as Molecular Chaperones to Alleviate Stress-induced Damage

“…In fact, unlike LAP-A, where a majority of substitutions at Lys-354 cause hexamer disassembly, none of the LAP-N Lys-357 mutants disrupted the LAP-N hexamer. The sensitivity of the LAP-N chaperone to mutations in a catalytic residue may be consistent with the facts that LAP-N is less stable in vitro than LAP-A (14) and the peptidase substrate specificity of LAP-N is distinct from that of LAP-A (14,30,34,35).…”

“…Currently, no plant aminopeptidase is known to function as a molecular chaperone. However, the tomato LAP-A has several compelling biochemical characteristics that are shared with molecular chaperones, including its stability, high temperature optimum (60 -70°C), high pH optimum (9.0), and induction by a wide range of stresses (16,30). Finally, two microbial proteins are known to possess both chaperone and aminopeptidase activity in vitro: the E. coli Hsp31 chaperone and a Shizosaccharomyes pombe aspartyl aminopeptidase (25,31).…”

Isolation and Characterization of the Neutral Leucine Aminopeptidase (LapN) of Tomato

“…The overexpression of an Arabidopsis LAP1-fusion protein in E. coli assembled into a stable homohexameric enzyme, although a high mass complex was also identified. AtLAP1's ability to hydrolyze Leu-␤-NAP has not been tested; however, using the Leu-pnitroanilide substrate (AtLAP1) had a specific activity similar to that reported for the tomato LAP-A1 and potato LAP (Bartling and Nosek, 1994;Herbers et al, 1994;Gu et al, 1999;Gu and Walling, 2000).…”

“…Aminopeptidase activity assays using nine amino-acyl-␤-NAP subtrates (Sigma) were performed according to the methods described by Gu et al (1999). Two micrograms of purified His 6 -LAP-N and His 6 -LAP-A enzymes was assayed per reaction.…”

Leucine aminopeptidase of the human blood flukes, Schistosoma mansoni and Schistosoma japonicum

“…In this context, it is of interest to note that cathepsin L1 (Dalton et al, 1996;Brady et al, 1999a,b) and cathepsin B2 (Caffrey et al, 2002) endoproteases have also been localised to the tegument and hence it is plausible that a multi-enzymatic degradation process, similar to that described for hemoglobin digestion in the gut, also occurs in this tissue. The up-regulation of tomato LAP expression is associated with mechanical wounding or infestation and may be part of a protein turnover or tissue repair mechanism (Gu et al, 1999). Given that the schistosome surface membrane is in constant flux, it is possible that cathepsin L1, cathepsin B2 and LAP work in concert in the dismantling, degradation and turnover of the protein components of the membrane.…”