First published online July 9, 2004; 10.1104/pp.104.043745
Plant Physiology 135:1221-1230 (2004)
© 2004 American Society of Plant Biologists
BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES
The Biosynthesis of D-Galacturonate in Plants. Functional Cloning and Characterization of a Membrane-Anchored UDP-D-Glucuronate 4-Epimerase from Arabidopsis1
Michael Mølhøj2,
Rajeev Verma and
Wolf-Dieter Reiter*
Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06269
Pectic cell wall polysaccharides owe their high negative charge to the presence of D-galacturonate, a monosaccharide that appears to be present only in plants and some prokaryotes. UDP-D-galacturonate, the activated form of this sugar, is known to be formed by the 4-epimerization of UDP-D-glucuronate; however, no coding regions for the epimerase catalyzing this reaction have previously been described in plants. To better understand the mechanisms by which precursors for pectin synthesis are produced, we used a bioinformatics approach to identify and functionally express a UDP-D-glucuronate 4-epimerase (GAE1) from Arabidopsis. GAE1 is predicted to be a type II membrane protein that belongs to the family of short-chain dehydrogenases/reductases. The recombinant enzyme expressed in Pichia pastoris established a 1.3:1 equilibrium between UDP-D-galacturonate and UDP-D-glucuronate but did not epimerize UDP-D-Glc or UDP-D-Xyl. Enzyme assays on cell extracts localized total UDP-D-glucuronate 4-epimerase and recombinant GAE1 activity exclusively to the microsomal fractions of Arabidopsis and Pichia, respectively. GAE1 had a pH optimum of 7.6 and an apparent Km of 0.19 mM. The recombinant enzyme was strongly inhibited by UDP-D-Xyl but not by UDP, UDP-D-Glc, or UDP-D-Gal. Analysis of Arabidopsis plants transformed with a GAE1:GUS construct showed expression in all tissues. The Arabidopsis genome contains five GAE1 paralogs, all of which are transcribed and predicted to contain a membrane anchor. This suggests that all of these enzymes are targeted to an endomembrane system such as the Golgi where they may provide UDP-D-galacturonate to glycosyltransferases in pectin synthesis.
1 This work was supported by the U.S. Department of Energy (grant no. DE–FG02–95ER20203) and by a fellowship from the Danish Agricultural and Veterinary Research Council (grant no. SJVF 23000237 to M.M.).
2 Present address: Micromet AG, Staffelseestr. 2, 81477 Munich, Germany.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.043745.
* Corresponding author; e-mail wdreiter{at}uconnvm.uconn.edu; fax 860–486–4331.
Received March 30, 2004;
returned for revision April 21, 2004;
accepted April 21, 2004.
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