Bifunctional cytosolic UDP-glucose 4-epimerases catalyse the interconversion between UDP-D-xylose and UDP-L-arabinose in plants.
UDP-sugars serve as substrates in the synthesis of cell wall polysaccharides and are themselves generated through sequential interconversion reactions from UDP-Glc (UDP-glucose) as the starting substrate in the cytosol and the Golgi apparatus. For the present study, a soluble enzyme with UDP-Xyl (UDP-xylose) 4-epimerase activity was purified approx. 300-fold from pea (Pisum sativum L.) sprouts by conventional chromatography. The N-terminal amino acid sequence of the enzyme revealed that it is encoded by a predicted UDP-Glc 4-epimerase gene, PsUGE1, and is distinct from the UDP-Xyl 4-epimerase localized in the Golgi apparatus. rPsUGE1 (recombinant P. sativum UGE1) expressed in Escherichia coli exhibited both UDP-Xyl 4-epimerase and UDP-Glc 4-epimerase activities with apparent Km values of 0.31, 0.29, 0.16 and 0.15 mM for UDP-Glc, UDP-Gal (UDP-galactose), UDP-Ara (UDP-L-arabinose) and UDP-Xyl respectively. The apparent equilibrium constant for UDP-Ara formation from UDP-Xyl was 0.89, whereas that for UDP-Gal formation from UDP-Glc was 0.24. Phylogenetic analysis revealed that PsUGE1 forms a group with Arabidopsis UDP-Glc 4-epimerases, AtUGE1 and AtUGE3, apart from a group including AtUGE2, AtUGE4 and AtUGE5. Similar to rPsUGE1, recombinant AtUGE1 and AtUGE3 expressed in E. coli showed high UDP-Xyl 4-epimerase activity in addition to their UDP-Glc 4-epimerase activity. Our results suggest that PsUGE1 and its close homologues catalyse the interconversion between UDP-Xyl and UDP-Ara as the last step in the cytosolic de novo pathway for UDP-Ara generation. Alternatively, the net flux of metabolites may be from UDP-Ara to UDP-Xyl as part of the salvage pathway for Ara.
Kotake
Toshihisa
T
Division of Life Science, Graduate School of Science and Engineering, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama 338-8570, Japan. kotake@molbiol.saitama-u.ac.jp
Takata
Ryohei
R
Verma
Rajeev
R
Takaba
Masato
M
Yamaguchi
Daisuke
D
Orita
Takahiro
T
Kaneko
Satoshi
S
Matsuoka
Koji
K
Koyama
Tetsuo
T
Reiter
Wolf-Dieter
WD
Tsumuraya
Yoichi
Y
D000595:Amino Acid Sequence; D000818:Animals; D055162:Biocatalysis; D017124:Conserved Sequence; D003600:Cytosol / Q000201:enzymology*; D006801:Humans; D008969:Molecular Sequence Data; D018532:Pisum sativum / Q000201:enzymology*; D010802:Phylogeny; D036226:Seedlings / Q000201:enzymology; D016415:Sequence Alignment; D013379:Substrate Specificity; D014534:UDPglucose 4-Epimerase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D014539:Uridine Diphosphate Sugars / Q000378:metabolism*; D014540:Uridine Diphosphate Xylose / Q000378:metabolism*
D014539:Uridine Diphosphate Sugars; C040817:uridine diphosphate arabinose; D014540:Uridine Diphosphate Xylose; D014534:UDPglucose 4-Epimerase