Biochem. J. (2006) 394
(115–124) (Printed in Great Britain)
Gene expression patterns and catalytic properties of UDP-D-glucose 4-epimerases from barley (Hordeum vulgare L.)
Qisen ZHANG, Maria HRMOVA, Neil J. SHIRLEY, Jelle LAHNSTEIN and Geoffrey B. FINCHER1
Australian Centre for Plant Functional Genomics, School of Agriculture and Wine, University of Adelaide, Waite Campus, Glen Osmond, SA 5064, Australia
UGE (UDP-Glc 4-epimerase or UDP-Gal 4-epimerase; EC 5.1.3.2) catalyses the interconversion of UDP-Gal and UDP-Glc. Both nucleotide sugars act as activated sugar donors for the biosynthesis of cell wall polysaccharides such as cellulose, xyloglucans, (1,3;1,4)-b-D-glucan and pectins, together with other biologically significant compounds including glycoproteins and glycolipids. Three members of the HvUGE (barley UGE) gene family, designated HvUGE1, HvUGE2 and HvUGE3, have been characterized. Q-PCR (quantitative real-time PCR) showed that HvUGE1 mRNA was most abundant in leaf tips and mature roots, but its expression levels were relatively low in basal leaves and root tips. The HvUGE2 gene was transcribed at significant levels in all organs examined, while HvUGE3 mRNA levels were very low in all the organs. Heterologous expression of a near full-length cDNA confirmed that HvUGE1 encodes a functional UGE. A non-covalently bound NAD+ was released from the enzyme after denaturing with aqueous ethanol and was identified by its spectrophotometric properties and by electrospray ionization MS. The Km values were 40 µM for UDP-Gal and 55 µM for UDP-Glc. HvUGE also catalyses the interconversion of UDP-GalNAc and UDP-GlcNAc, although it is not known if this has any biological significance. A three-dimensional model of the HvUGE revealed that its overall structural fold is highly conserved compared with the human UGE and provides a structural rationale for its ability to bind UDP-GlcNAc.
Key words: barley, gene expression, nucleotide sugar, plant cell wall, UDP-D-glucose 4-epimerase (UGE), UDP-GlcNAc.
Abbreviations used: EPS I, exopolysaccharide I; EST, expressed sequence tag; UGE, UDP-Glc 4-epimerase or UDP-Gal 4-epimerase; HvUGE, barley UGE; IPTG, isopropyl b-D-thiogalactoside; Ni-NTA, Ni2+-nitrilotriacetate; Q-PCR, quantitative real-time PCR; QTL, quantitative trait locus; R.M.S.D., root mean square deviation; UXS, UDP-GlcA decarboxylase; HvUGAE, barley UDP-GlcA 4-epimerase; HvUGDH, barley UDP-Glc dehydrogenase.
1To whom correspondence should be addressed (email geoff.fincher@adelaide.edu.au).
The nucleotide sequence data reported will appear in DDBJ, EMBL, GenBank® and GSDB Nucleotide Sequence Databases under the accession numbers AY943955, AY943956 and AY943954.
Received 15 August 2005/1 November 2005; accepted 3 November 2005
Published as BJ Immediate Publication 3 November 2005, doi:10.1042/BJ20051329
The Biochemical Society, London ©2006
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