RRC ID 3078
Author Oka T, Nemoto T, Jigami Y.
Title Functional analysis of Arabidopsis thaliana RHM2/MUM4, a multidomain protein involved in UDP-D-glucose to UDP-L-rhamnose conversion.
Journal J. Biol. Chem.
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.
Volume 282(8)
Pages 5389-403
Published 2007-2-23
DOI 10.1074/jbc.M610196200
PII M610196200
PMID 17190829
MeSH 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
IF 4.106
Times Cited 79
WOS Category BIOCHEMISTRY & MOLECULAR BIOLOGY
Resource
Arabidopsis / Cultured plant cells, genes pda08705