Document Detail


Functional analysis of Arabidopsis thaliana RHM2/MUM4, a multidomain protein involved in UDP-D-glucose to UDP-L-rhamnose conversion.
MedLine Citation:
PMID:  17190829     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Takuji Oka; Tadashi Nemoto; Yoshifumi Jigami
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2006-12-26
Journal Detail:
Title:  The Journal of biological chemistry     Volume:  282     ISSN:  0021-9258     ISO Abbreviation:  J. Biol. Chem.     Publication Date:  2007 Feb 
Date Detail:
Created Date:  2007-02-19     Completed Date:  2007-04-17     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  2985121R     Medline TA:  J Biol Chem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  5389-403     Citation Subset:  IM    
Affiliation:
Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki 305-8566, Japan.
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MeSH Terms
Descriptor/Qualifier:
Amino Acid Motifs / genetics
Arabidopsis / chemistry,  enzymology*
Arabidopsis Proteins / chemistry,  genetics,  metabolism*
Cell Wall / metabolism*
Genome, Plant / physiology*
Multienzyme Complexes / chemistry,  genetics,  metabolism*
Mutagenesis, Site-Directed
Pectins / biosynthesis,  chemistry,  genetics
Rhamnose / chemistry,  genetics,  metabolism*
Saccharomyces cerevisiae / genetics
Seeds / chemistry,  enzymology,  genetics
Uridine Diphosphate Glucose / chemistry,  genetics,  metabolism*
Uridine Diphosphate Xylose / chemistry,  genetics,  metabolism
Chemical
Reg. No./Substance:
0/Arabidopsis Proteins; 0/MUM4 protein, Arabidopsis; 0/Multienzyme Complexes; 0/Pectins; 0/rhamnogalacturonan I; 0/rhamnogalacturonan II; 10485-94-6/Rhamnose; 133-89-1/Uridine Diphosphate Glucose; 3616-06-6/Uridine Diphosphate Xylose

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