The genetics, transcriptional profiles, and catalytic properties of UDP-alpha-D-xylose 4-epimerases from barley.
Cell walls in the grasses contain relatively high levels of heteroxylans and, in particular, arabinoxylans. Enzymes and corresponding genes that are involved in the provision of sugar nucleotide substrates represent potential control points for arabinoxylan biosynthesis. Following expressed sequence tag database analyses, three genes encoding barley (Hordeum vulgare) UDP-d-xylose 4-epimerases (UXE; EC 5.1.3.5), designated HvUXE1, HvUXE2, and HvUXE3, were cloned and their positions on genetic maps defined. To confirm the identity of the genes, a cDNA construct encoding HvUXE1 was expressed in Pichia pastoris. The purified, recombinant HvUXE1 catalyzed the freely reversible interconversion of UDP-alpha-d-xylopyranose and UDP-beta-l-arabinopyranose, with K(m) values of 1.8 and 1.4 mm, respectively. At equilibrium, the ratio of substrate to product was approximately 1:1. Each molecule of heterologously expressed HvUXE1 enzyme contained about one molecule of noncovalently bound NAD(+). Molecular modeling provided a structural rationale for the substrate specificity of the UDP-d-xylose 4-epimerase and, in particular, explained its tight specificity for UDP-xylose compared with other sugar nucleotide epimerases. Quantitative transcript analyses performed for each of the three genes in a range of organs showed, inter alia, that in developing barley endosperm HvUXE1 and HvUXE3 mRNA levels peaked at a time when UDP-alpha-d-xylopyranose synthase (UXS) transcripts also reached a maximum and when arabinoxylan biosynthesis was initiated. Furthermore, the data revealed that the transcription of HvUXE and HvUXS gene family members is coordinated with the incorporation of pentose sugars onto cell walls in barley leaves, roots, and developing endosperm.
Zhang
Qisen
Q
Australian Centre for Plant Functional Genomics, School of Agriculture, Food, and Wine, University of Adelaide, Waite Campus, Glen Osmond, South Australia 5064, Australia.
Shirley
Neil J
NJ
Burton
Rachel A
RA
Lahnstein
Jelle
J
Hrmova
Maria
M
Fincher
Geoffrey B
GB
D000595:Amino Acid Sequence; D002238:Carbohydrate Epimerases / Q000235:genetics / Q000378:metabolism*; D002473:Cell Wall / Q000737:chemistry; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D056625:Endosperm / Q000737:chemistry; D020869:Gene Expression Profiling; D018506:Gene Expression Regulation, Plant; D001467:Hordeum / Q000201:enzymology / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010802:Phylogeny; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D018749:RNA, Plant / Q000235:genetics; D016415:Sequence Alignment; D013379:Substrate Specificity; D014539:Uridine Diphosphate Sugars / Q000378:metabolism; D014994:Xylose / Q000031:analogs & derivatives / Q000378:metabolism
D010940:Plant Proteins; D018749:RNA, Plant; D014539:Uridine Diphosphate Sugars; C431715:xylopyranose; C040817:uridine diphosphate arabinose; D014994:Xylose; D002238:Carbohydrate Epimerases