The biosynthesis of D-Galacturonate in plants. functional cloning and characterization of a membrane-anchored UDP-D-Glucuronate 4-epimerase from Arabidopsis.
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.
Mølhøj
Michael
M
Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06269, USA.
Verma
Rajeev
R
Reiter
Wolf-Dieter
WD
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics*; D002238:Carbohydrate Epimerases / Q000737:chemistry / Q000235:genetics*; D003001:Cloning, Molecular; D004795:Enzyme Stability; D035264:Flowers / Q000201:enzymology; D005690:Galactose / Q000096:biosynthesis*; D006603:Hexuronic Acids / Q000378:metabolism*; D008969:Molecular Sequence Data; D018515:Plant Leaves / Q000201:enzymology; D018517:Plant Roots / Q000201:enzymology
D029681:Arabidopsis Proteins; D006603:Hexuronic Acids; C007819:galacturonic acid; D002238:Carbohydrate Epimerases; C023258:UDPglucuronate 4-epimerase; D005690:Galactose