The Genetics, Transcriptional Profiles, and Catalytic Properties of UDP-α-d-Xylose 4-Epimerases from Barley  

Abstract

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-a-D-xylopyranose and UDP-b-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-a-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.

Infant food applications of complex carbohydrates: Structure, synthesis, and function

“…[171] Arabinoxylan biosynthesis is believed to occur by the action of a xylopyranose synthase and arabinose synthase. [175] Glucomannan ( 34 ) is produced by a glucomannan synthase complex. [176] Finally, the backbone of xyloglucans is synthesized by cellulose synthase-like proteins, and the side chains are added with the aid of the corresponding glycosyltransferases.…”

Genetics, Transcriptional Profiles, and Catalytic Properties of the UDP-Arabinose Mutase Family from Barley

“…Total RNA preparations from developing endosperms, leaves, roots, coleoptiles, and other tissue types were extracted as described by Zhang et al 27 and converted to cDNA as described by Burton et al 32 . Levels of barley HvUAM mRNA were determined by quantitative Q-PCR in a RG 2000 Rotor-Gene Real Time Thermal Cycler (Sydney, Australia) according to Zhang et al 27 . Primer pairs for four members of the HvUAM gene family were designed and are listed in Table 3.…”

Plant Nucleotide Sugar Formation, Interconversion, and Salvage by Sugar Recycling*

“…A second isoform (UXE2, At2g34850) that has at least 76% sequence identity to UXE1 has been identified in Arabidopsis (19). There are two UXE isoforms in rice and three UXE isoforms in barley (176).…”