Identification of a Ca(2+)-pectate binding site on an apoplastic peroxidase.
An apoplastic isoperoxidase from zucchini (APRX) was shown to bind strongly to polygalacturonic acid in their Ca(2)+-induced conformation. By homology modeling, we were able to identify a motif of four clustered arginines (positions 117, 262, 268, and 271) that could be responsible for this binding. To verify the role of these arginine residues in the binding process, we prepared three mutants of APRX (M1, R117S; M2, R262Q/R268S; and M3, R262Q/R268S/R271Q). APRX and the three mutants were expressed as recombinant glycoproteins by the baculovirus-insect cell system. This procedure yielded four active enzymes with similar molecular masses that were tested for their ability to bind Ca(2)+-pectate. Recombinant wild-type APRX exhibited an affinity for the pectic structure comparable to that of the native plant isoperoxidase. The mutations impaired binding depending on the number of arginine residues that were replaced. M1 and M2 showed intermediate affinities, whereas M3 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 APRX bears a Ca(2)+-pectate binding site formed by four clustered arginines. This site could ensure that APRX is properly positioned in cell walls, using unesterified domains of pectins as a scaffold.
Carpin
S
S
Laboratoire de Biochimie et Physiologie Végétales, Université de Genève, Place de l'Université 3, 1211 Genève 4, Switzerland.
Crèvecoeur
M
M
de Meyer
M
M
Simon
P
P
Greppin
H
H
Penel
C
C
D001120:Arginine / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D002473:Cell Wall / Q000378:metabolism; D019662:Cucurbitaceae / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D004586:Electrophoresis; D018546:Hypocotyl / Q000166:cytology; D008958:Models, Molecular; D009154:Mutation; D010368:Pectins / Q000378:metabolism*; D009195:Peroxidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010544:Peroxidases / Q000378:metabolism; D011487:Protein Conformation; D055672:Static Electricity
D002135:Calcium-Binding Proteins; D010368:Pectins; D001120:Arginine; D010544:Peroxidases; D009195:Peroxidase; C120494:anionic peroxidase; C003181:polygalacturonic acid