The biosynthesis of D-galacturonate in plants. Functional cloning and characterization of a membrane-anchored UDP-D-Glucuronate 4-epimerase from Arabidopsis

被引:78
作者
Molhoj, M [1 ]
Verma, R [1 ]
Reiter, WD [1 ]
机构
[1] Univ Connecticut, Dept Mol & Cell Biol, Storrs, CT 06269 USA
关键词
D O I
10.1104/pp.104.043745
中图分类号
Q94 [植物学];
学科分类号
071001 ;
摘要
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 K-m of 0.19 mm. The recombinant enzyme was strongly inhibited by UDP-D-Xvl but not by UDF, 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.
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页码:1221 / 1230
页数:10
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