Functional analysis of Arabidopsis thaliana RHM2/MUM4, a multidomain protein involved in UDP-D-glucose to UDP-L-rhamnose conversion.
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.
Oka
Takuji
T
Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki 305-8566, Japan.
Nemoto
Tadashi
T
Jigami
Yoshifumi
Y
D020816:Amino Acid Motifs / Q000235:genetics; D017360:Arabidopsis / Q000737:chemistry / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002473:Cell Wall / Q000378:metabolism*; D018745:Genome, Plant / Q000502:physiology*; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010368:Pectins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012210:Rhamnose / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012639:Seeds / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D014532:Uridine Diphosphate Glucose / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014540:Uridine Diphosphate Xylose / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D029681:Arabidopsis Proteins; C518819:MUM4 protein, Arabidopsis; D009097:Multienzyme Complexes; C042491:rhamnogalacturonan I; C042492:rhamnogalacturonan II; D014540:Uridine Diphosphate Xylose; D010368:Pectins; D012210:Rhamnose; D014532:Uridine Diphosphate Glucose