Functional analysis of Arabidopsis thaliana RHM2/MUM4, a multidomain protein involved in UDP-D-glucose to UDP-L-rhamnose conversion.

摘要: UDP-L-rhamnose is required for the biosynthesis of cell wall rhamnogalacturonan-I, rhamnogalacturonan-II, and natural compounds in plants. It has been suggested that RHM2/MUM4 gene involved conversion UDP-D-glucose to on basis its effect rhamnogalacturonan-I-directed development Arabidopsis thaliana. RHM2/MUM4-related genes, RHM1 RHM3, can be found A. thaliana genome. Here we present direct evidence all three RHM proteins have 4,6-dehydratase, UDP-4-keto-6-deoxy-D-glucose 3,5-epimerase, UDP-4-keto-L-rhamnose 4-keto-reductase activities cytoplasm when expressed yeast Saccharomyces cerevisiae. Functional domain analysis revealed N-terminal region RHM2 (RHM2-N; amino acids 1-370) first activity C-terminal (RHM2-C; 371-667) two following activities. This suggests converts UDP-d-glucose via an intermediate. Site-directed mutagenesis mucilage defects MUM4-1 MUM4-2 mutant seeds are caused by abolishment enzymatic strains furthermore, GXXGXX(G/A) YXXXK motifs important activity. Moreover, a kinetic purified His(6)-tagged RHM2-N protein 5.9-fold higher affinity than dTDP-D-glucose, preferred substrate dTDP-D-glucose 4,6-dehydratase from bacteria. strongly inhibited UDP-L-rhamnose, UDP-D-xylose, UDP but not other sugar nucleotides, suggesting maintains cytoplasmic levels feedback inhibition UDP-D-xylose.