First published online March 12, 2004; 10.1104/pp.103.037192
Plant Physiology 134:1337-1346 (2004)
© 2004 American Society of Plant Biologists
BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES
A Bifunctional 3,5-Epimerase/4-Keto Reductase for Nucleotide-Rhamnose Synthesis in Arabidopsis1
Gregory Watt,
Christine Leoff,
April D. Harper and
Maor Bar-Peled*
Complex Carbohydrate Research Center and Department of Plant Biology, University of Georgia, Athens, Georgia 30602–4712
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- -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--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°C. The apparent Km for NADPH is 90 µM and 16.9 µM for dTDP-4-keto-6-deoxy-Glc. The Arabidopsis enzyme can also form UDP--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.
1 This work was supported in part by the U.S. Department of Agriculture (grant no. 2002–35318–12620 to M.B.-P.) and by the U.S. Department of Energy (center grant no. DE–FG05–93ER20097) for the DOE Center for Plant and Microbial Complex Carbohydrates.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.037192.
* Corresponding author; e-mail peled{at}ccrc.uga.edu; fax 706–542–4412.
Received December 3, 2003;
returned for revision December 23, 2003;
accepted December 23, 2003.
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