Analysis of the structure and function of cAAP, a chloroplast-localized serine peptidase using site directed mutagenesis of the predicted dimerization domain

The structure and function of the prolyl oligopeptidase family have been studied intensely. The family includes aclyaminoacyl peptidase. Arabidopsis thaliana gene At5g36210 encodes a chloroplast-localized aclyaminoacyl peptidase (cAAP). Several acylaminoacyl peptidases form dimers or higher order complexes. Although little is known about the structure and function of cAAP, the structure of the archaeon ortholog from Aeropyrum pernix (apAPH), has been determined. The proposed dimerization interface residues were determined from threading the cAAP with the archaeon ortholog, and these residues were subjected to mutagenesis as well as the catalytic serine. Plasmid constructs with mutated versions of cAAP were introduced into the caap mutant in order to isolate lines that expressed high levels of each mutant protease. Activity analyses were used to determine the effects of mutation. A difference in activity was not observed, except for the mutation of the catalytic serine. Wild type overexpression samples suggests a monomeric protein. The study did not provide data to suggest an oligomeric state of cAAP, and thus based on the current study, cAAP likely is a monomer in Arabidopsis. The study suggest that the constructs were transported into the stroma, but construction of chimeric proteins using Rubisco small subunit chloroplast leader and an epitope tag for easier identification and may increase stromal transport efficiency. Substrate specificity studies may shed light on the activity analysis. The combination of future studies may ascertain the dimerization structure and may shed light on the function of cAAP.