Identification of a Ca2+-pectate binding site on an apoplastic peroxidase

An apoplastic isoperoxidase from zucchini (APRX) was shown to bind strongly to polygalacturonic acid in their Ca2+-induced conformation. By homology m odeling, we were able to identify a motif of four clustered arginines (posi tions 117, 262, 268, and 271) that could be responsible for this binding. T o verify the role of these arginine residues in the binding process, we pre pared three mutants of APRX (MI, R117S; M2, R262Q/R268S; and M3, R262Q/R268 S/R271Q). APRX and the three mutants were expressed as recombinant glycopro teins by the baculovirus-insect cell system, This procedure yielded four ac tive enzymes with similar molecular masses that were tested for their abili ty to bind Ca2+-pectate. Recombinant wild-type APRX exhibited an affinity f or the pectic structure comparable to that of the native plant isoperoxidas e. The mutations impaired binding depending on the number of arginine resid ues that were replaced. M1 and M2 showed intermediate affinities, whereas M 3 did not bind at all, This was demonstrated using an in vitro binding test and on cell walls of hypocotyl cross-sections. It can be concluded that AP RX bears a Ca2+-pectate binding site formed by four clustered arginines. Th is site could ensure that APRX is properly positioned in cell walls, using unesterified domains of pectins as a scaffold.