A bifunctional 3,5-epimerase/4-keto reductase for nucleotide-rhamnose synthesis in Arabidopsis.
l-Rhamnose is a component of plant cell wall pectic polysaccharides, diverse secondary metabolites, and some glycoproteins. The biosynthesis of the activated nucleotide-sugar form(s) of rhamnose utilized by the various rhamnosyltransferases is still elusive, and no plant enzymes involved in their synthesis have been purified. In contrast, two genes (rmlC and rmlD) have been identified in bacteria and shown to encode a 3,5-epimerase and a 4-keto reductase that together convert dTDP-4-keto-6-deoxy-Glc to dTDP-beta-l-rhamnose. We have identified an Arabidopsis cDNA that contains domains that share similarity to both reductase and epimerase. The Arabidopsis gene encodes a protein with a predicated molecular mass of approximately 33.5 kD that is transcribed in all tissue examined. The Arabidopsis protein expressed in, and purified from, Escherichia coli converts dTDP-4-keto-6-deoxy-Glc to dTDP-beta-l-rhamnose in the presence of NADPH. These results suggest that a single plant enzyme has both the 3,5-epimerase and 4-keto reductase activities. The enzyme has maximum activity between pH 5.5 and 7.5 at 30 degrees C. The apparent K(m) for NADPH is 90 microm and 16.9 microm for dTDP-4-keto-6-deoxy-Glc. The Arabidopsis enzyme can also form UDP-beta-l-rhamnose. To our knowledge, this is the first example of a bifunctional plant enzyme involved in sugar nucleotide synthesis where a single polypeptide exhibits the same activities as two separate prokaryotic enzymes.
Watt
Gregory
G
Complex Carbohydrate Research Center and Department of Plant Biology, University of Georgia, Athens, Georgia 30602-4712, USA.
Leoff
Christine
C
Harper
April D
AD
Bar-Peled
Maor
M
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism; D002238:Carbohydrate Epimerases / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D007658:Ketone Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D012210:Rhamnose / Q000096:biosynthesis*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D029681:Arabidopsis Proteins; D018076:DNA, Complementary; D007658:Ketone Oxidoreductases; D002238:Carbohydrate Epimerases; C488385:dTDP-rhamnose synthase, Arabidopsis; D012210:Rhamnose