A Bifunctional 3,5-Epimerase/4-Keto Reductase for Nucleotide-Rhamnose Synthesis in Arabidopsis

Abstract

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-b-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-b-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 308C. The apparent K m for NADPH is 90 mM and 16.9 mM for dTDP-4-keto-6-deoxy-Glc. The Arabidopsis enzyme can also form UDP-b-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.L-Rhamnose is a component of the plant cell wall pectic polysaccharides rhamnogalacturonan I (RG-I) and rhamnogalacturonan II (RG-II; Ridley et al., 2001) and is also present in diverse secondary metabolites including anthocyanins, flavonoids, and triterpenoids (Das et al., 1987;Bar-Peled et al., 1991;van Setten et al., 1995;Shinozaki et al., 1996;Markham et al., 2000), in certain types of plant glycoproteins (Haruko and Haruko, 1999), and in arabinogalactan proteins (Pellerin et al., 1995). The specific enzymes that attach rhamnose to each molecule are known as rhamnosyltransferases (RhaTs). To date, only a small number of RhaTs have been studied, and those were involved in flavonoid rhamnosylation. The characterized RhaTs utilize UDP-b-L-rhamnose (UDP-b-L-Rha) as the donor substrate (Kamsteeg et al., 1978;Feingold, 1982;Bar-Peled et al., 1991), although in mung bean (Vigna radiata), both dTDP-b-L-rhamnose (dTDP-b-L-Rha) and UDP-b-L-Rha were reported to act as sugar donors for the rhamnosylation of flavonoids (Barber and Neufeld, 1961).We are studying the enzymes involved in the synthesis of the nucleotide-rhamnose as part of our effort to understand the synthesis of pectic polysaccharides. To date, the rhamnosylation of plant polysaccharides and glycoproteins has not been studied. Thus, the identity of the activated form(s) of rhamnose needed for the synthesis of these macromolecules is not known with certainty. The enzymes required for the synthesis of the activated form(s) of rhamnose in plants have also not been purified.In contrast, much more is known about the synthesis of rhamnose in...

Functional identification of an Arabidopsis pectin biosynthetic homogalacturonan galacturonosyltransferase

“…A complete understanding of pectin function requires knowledge of pectin biosynthetic enzymes and their corresponding genes. Although the enzyme activities of proteins encoded by some pectin biosynthetic genes, particularly those involved in the synthesis of sugar nucleotides, have been elucidated (5)(6)(7)(8)(9), the activities of putative pectin biosynthetic glycosyltransferases [e.g., QUA1 (10) and NpGUT1 (11)] have not been definitively demonstrated.…”

Biosynthesis of UDP-4-keto-6-deoxyglucose and UDP-rhamnose in Pathogenic Fungi Magnaporthe grisea and Botryotinia fuckeliana

“…The M. grisea homolog shares 37% amino acid sequence identity to a functional bacterial rmlB, dTDP-Glc 4,6-dehydratase (25,26), from Salmonella, Streptococcus, and 42% identity to the N-terminal portion of plant URS, Rhm (supplemental Fig. S3A) (18,19). Such sequence identity was not sufficient to establish the function of the gene or determine whether the encoded protein utilizes dTDP-Glc or UDP-Glc.…”

“…A bi-functional 3,5-epimerase and 4-reductase (U4k6dG-ER) converts U4k6dG to UDP-Rha and NADP ϩ . In plants (18,19) one enzyme has 4,6-dehydratase, 3,5-epimerase, and 4-reductase activities to convert UDP-Glc to UDP-Rha.…”

“…An enzyme with 4-reductase activity (rmlD) converts the keto-rhamnose to dTDP-Rha (15)(16)(17). Plants have a single protein (Rhm (UDP-L-rhamnose synthase) also annotated as URS) that converts UDP-Glc to UDP-Rha (18,19). In addition, plants have a bi-functional protein (nucleotide-rhamnose synthase/epimerase-reductase (NRS/ER)) that has 3,5-epimerization and 4-reduction activities (19).…”

LASSO modeling of the Arabidopsis thaliana seed/seedling transcriptome: a model case for detection of novel mucilage and pectin metabolism genes

“…55 Furthermore this list included NRS/ER, a gene which shows strong similarity to RHM2 but lacks one of its domains and is thus likely to be involved in the synthesis of UDP-L-rhamnose as well. 56 In addition, the model contained AtNST-KT1 (At4g39390), a gene which represents a monospecific nucleotide sugar transporter. 57 Specifically it was shown that this transporter exchanges UDP-Gal for UMP and is localized to Golgi membranes.…”