Biochemical characterization of the kinase domain of the rice disease resistance receptor-like kinase XA21.
The rice disease resistance gene, Xa21, encodes a receptor kinase-like protein consisting of leucine-rich repeats in the putative extracellular domain and a serine/threonine kinase in the putative intracellular domain. The putative XA21 kinase domain was expressed as maltose-binding and glutathione S-transferase fusion proteins in Escherichia coli. The fusion proteins are capable of autophosphorylation. Phosphoamino acid analysis of the glutathione S-transferase fusion protein indicates that only serine and threonine residues are phosphorylated. The relative phosphorylation rate of the XA21 kinase against increasing enzyme concentrations follows a first-order rather than second-order kinetics, indicating an intramolecular phosphorylation mechanism. Moreover, the active XA21 kinase cannot phosphorylate a kinase-deficient mutant of XA21 kinase. The enzymatic activity of the XA21 kinase in a buffer containing Mn(2+) is at least 15 times higher than that with Mg(2+). The K(m) and V(max) of XA21 kinase for ATP are 0.3 microm and 8.4 nmol/mg/min, respectively. Tryptic phosphopeptide mapping reveals that multiple sites on the XA21 kinase are phosphorylated. Finally, our data suggest that the region of XA21 kinase corresponding to the RD kinase activation domain is not phosphorylated, revealing a distinct mode of action compared with the tomato Pto serine/threonine kinase conferring disease resistance.
Liu
Guo-Zhen
GZ
Department of Plant Pathology, University of Florida, Gainesville, Florida 32611, USA.
Pi
Li-Ya
LY
Walker
John C
JC
Ronald
Pamela C
PC
Song
Wen-Yuan
WY
D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D007700:Kinetics; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010766:Phosphorylation; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000378:metabolism; D013912:Threonine / Q000737:chemistry / Q000378:metabolism
D017931:DNA Primers; D010940:Plant Proteins; D011994:Recombinant Proteins; C097148:Xa21 protein, Oryza sativa; D013912:Threonine; D012694:Serine; D017346:Protein Serine-Threonine Kinases