PMID:17190829

Citation	Oka, T, Nemoto, T and Jigami, Y (2007) Functional analysis of Arabidopsis thaliana RHM2/MUM4, a multidomain protein involved in UDP-D-glucose to UDP-L-rhamnose conversion. J. Biol. Chem. 282:5389-403
Abstract	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 the RHM2/MUM4 gene is involved in conversion of UDP-D-glucose to UDP-L-rhamnose on the basis of its effect on rhamnogalacturonan-I-directed development in Arabidopsis thaliana. RHM2/MUM4-related genes, RHM1 and RHM3, can be found in the A. thaliana genome. Here we present direct evidence that all three RHM proteins have UDP-D-glucose 4,6-dehydratase, UDP-4-keto-6-deoxy-D-glucose 3,5-epimerase, and UDP-4-keto-L-rhamnose 4-keto-reductase activities in the cytoplasm when expressed in the yeast Saccharomyces cerevisiae. Functional domain analysis revealed that the N-terminal region of RHM2 (RHM2-N; amino acids 1-370) has the first activity and the C-terminal region of RHM2 (RHM2-C; amino acids 371-667) has the two following activities. This suggests that RHM2 converts UDP-d-glucose to UDP-L-rhamnose via an UDP-4-keto-6-deoxy-D-glucose intermediate. Site-directed mutagenesis of RHM2 revealed that mucilage defects in MUM4-1 and MUM4-2 mutant seeds of A. thaliana are caused by abolishment of RHM2 enzymatic activity in the mutant strains and furthermore, that the GXXGXX(G/A) and YXXXK motifs are important for enzymatic activity. Moreover, a kinetic analysis of purified His(6)-tagged RHM2-N protein revealed 5.9-fold higher affinity of RHM2 for UDP-D-glucose than for dTDP-D-glucose, the preferred substrate for dTDP-D-glucose 4,6-dehydratase from bacteria. RHM2-N activity is strongly inhibited by UDP-L-rhamnose, UDP-D-xylose, and UDP but not by other sugar nucleotides, suggesting that RHM2 maintains cytoplasmic levels of UDP-D-glucose and UDP-L-rhamnose via feedback inhibition by UDP-L-rhamnose and UDP-D-xylose.
Links	PubMed Online version:10.1074/jbc.M610196200
Keywords	Amino Acid Motifs/genetics; Arabidopsis/chemistry; Arabidopsis/enzymology; Arabidopsis Proteins/chemistry; Arabidopsis Proteins/genetics; Arabidopsis Proteins/metabolism; Cell Wall/metabolism; Genome, Plant/physiology; Multienzyme Complexes/chemistry; Multienzyme Complexes/genetics; Multienzyme Complexes/metabolism; Mutagenesis, Site-Directed; Pectins/biosynthesis; Pectins/chemistry; Pectins/genetics; Rhamnose/chemistry; Rhamnose/genetics; Rhamnose/metabolism; Saccharomyces cerevisiae/genetics; Seeds/chemistry; Seeds/enzymology; Seeds/genetics; Uridine Diphosphate Glucose/chemistry; Uridine Diphosphate Glucose/genetics; Uridine Diphosphate Glucose/metabolism; Uridine Diphosphate Xylose/chemistry; Uridine Diphosphate Xylose/genetics; Uridine Diphosphate Xylose/metabolism
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