The biosynthesis of the branched-chain sugar d-apiose in plants: functional cloning and characterization of a UDP-d-apiose/UDP-d-xylose synthase from Arabidopsis.
d-Apiose is a plant-specific branched-chain monosaccharide found in rhamnogalacturonan II (RG-II), apiogalacturonan, and several apioglycosides. Within RG-II, d-apiose serves as the binding site for borate, which leads to the formation of cross-links within the wall. Biochemical studies in duckweed and parsley have established that uridine 5'-diphospho-d-apiose (UDP-d-apiose) is formed from UDP-d-glucuronate by decarboxylation and re-arrangement of the carbon skeleton, leading to ring contraction and branch formation. The enzyme catalyzing this reaction also forms UDP-d-xylose by decarboxylation of UDP-d-glucuronate, and has therefore been named UDP-d-apiose/UDP-d-xylose synthase. Using a bioinformatics approach, we identified a candidate gene (AXS1) for this enzyme in Arabidopsis and functionally expressed its cDNA in Escherichia coli. The recombinant enzyme catalyzed the conversion of UDP-d-glucuronate to a mixture of UDP-d-apiose and UDP-d-xylose with a turnover number of 0.3 min-1. AXS1 required NAD+ for enzymatic activity, and was strongly inhibited by UDP-d-galacturonate. It was highly expressed in all plant organs consistent with a function in synthesizing an essential cell wall precursor. Database searches indicated the presence of closely related sequences in a variety of crop plants. The cloning of the AXS1 gene will help to investigate the biosynthesis of RG-II, and permit insights into the mechanism by which d-apiose and other branched monosaccharides are formed.
Mølhøj
Michael
M
Department of Molecular and Cell Biology, University of Connecticut, 75 North Eagleville Road, Storrs, CT 06269, USA.
Verma
Rajeev
R
Reiter
Wolf-Dieter
WD
D029681:Arabidopsis Proteins / Q000378:metabolism; D001483:Base Sequence; D002262:Carboxy-Lyases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D010429:Pentoses / Q000096:biosynthesis*; D010788:Photosynthesis / Q000502:physiology*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D029681:Arabidopsis Proteins; D017931:DNA Primers; D010429:Pentoses; D011994:Recombinant Proteins; C004983:apiose; D002262:Carboxy-Lyases; C023257:UDPglucuronate decarboxylase