Identification of residues critical for activity of the wound‐induced leucine aminopeptidase (LAP‐A) of tomato

Abstract

The importance of two putative Zn2+‐binding (Asp347, Glu429) and two catalytic (Arg431, Lys354) residues in the tomato leucine aminopeptidase (LAP‐A) function was tested. The impact of substitutions at these positions, corresponding to the bovine LAP residues Asp255, Glu334, Arg336, and Lys262, was evaluated in His6–LAP‐A fusion proteins expressed in Escherichia coli. Sixty‐five percent of the mutant His6–LAP‐A proteins were unstable or had complete or partial defects in hexamer assembly or stability. The activity of hexameric His6–LAP‐As on Xaa‐Leu and Leu‐Xaa dipeptides was tested. Most substitutions of Lys354 (a catalytic residue) resulted in His6–LAP‐As that cleaved dipeptides at slower rates. The Glu429 mutants (a Zn2+‐binding residue) had more diverse phenotypes. Some mutations abolished activity and others retained partial or complete activity. The E429D His6–LAP‐A enzyme had Km and kcat values similar to the wild‐type His6–LAP‐A. One catalytic (Arg431) and one Zn‐binding (Asp347) residue were essential for His6–LAP‐A activity, as most R431 and D347 mutant His6–LAP‐As did not hydrolyze dipeptides. The R431K His6–LAP‐A that retained the positive charge had partial activity as reflected in the 4.8‐fold decrease in kcat. Surprisingly, while the D347E mutant (that retained a negative charge at position 347) was inactive, the D347R mutant that introduced a positive charge retained partial activity. A model to explain these data is proposed.

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

“…Similar to LAP-A (Gu and Walling, 2002), the LAP-N had strong identity with microbial and animal LAPs in the 251-residue COOH domain, ranging from 42% (bovine LAP) to 48% (E. coli PepA) identity (data not shown). More recently, LAP genes were identified in two cyanobacteria, Nostoc sp.…”

“…The 55-kD LAP proteins with acidic pIs (LAP-As) accumulate in wounded leaves, whereas the 55-kD LAP proteins with neutral pIs (LAP-Ns) and the 66-and 77-kD LAP-like proteins are detected in both healthy and wounded tomato leaves. Comparisons of plant, animal, and microbe LAPs have shown that the COOH domain is highly conserved and harbors the two Zn 2ϩ -binding sites and catalytic domain of LAP, whereas N-terminal domains are diverged (Gu and Walling, 2002). Therefore, it was possible that antibodies recognizing the N-terminal domain of the LAP-A would discriminate the four classes of LAP-related polypeptides.…”

“…X-ray crystal structures of the bovine and E. coli LAPs have provided insight into the LAP catalytic mechanism (Kim and Lipscomb, 1994;Sträter and Lipscomb, 1995;Sträter et al, 1999a). The roles of selected residues of the E. coli and tomato LAPs in chromogenic or peptide substrate catalysis, respectively, have been tested by site-directed mutagenesis (Sträter et al, 1999b;Gu and Walling, 2002).In most plants, three classes of LAP-related polypeptides are detected using a tomato LAP antiserum, including the 66-and 77-kD LAP-like proteins and the 55-kD neutral LAP (LAP-N; Chao et al, 2000). Only in a subset of the Solanaceae is a second 55-kD LAP species (LAP-A) detected (Hildmann et al, 1992;Gu et al, 1996b;Chao et al, 2000).…”

“…X-ray crystal structures of the bovine and E. coli LAPs have provided insight into the LAP catalytic mechanism (Kim and Lipscomb, 1994;Sträter and Lipscomb, 1995;Sträter et al, 1999a). The roles of selected residues of the E. coli and tomato LAPs in chromogenic or peptide substrate catalysis, respectively, have been tested by site-directed mutagenesis (Sträter et al, 1999b;Gu and Walling, 2002).…”

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

“…Overexpression and Purification of LAP Proteins-The E. coli vectors that express the His 6 -LAP-A, His 6 -LAP-N, and the His 6 -LAP-A mutants (R431A, K354R, K354E, D347N, D347R, E429R, and E429V) were previously described (14,34). His 6 -LAP fusion proteins were expressed in and purified from E. coli according to Gu and Walling (35) with minor modifications.…”