The Biosynthesis of d-Galacturonate in Plants. Functional Cloning and Characterization of a Membrane-Anchored UDP-d-Glucuronate 4-Epimerase from Arabidopsis1

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American Society of Plant Biologists

RESUMO

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.

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