Molecular studies of galactan biosynthesis in red algae
Thesis (PhD)--Stellenbosch University, 2013.
ENGLISH ABSTRACT: Sulfated galactans (agarans and carrageenans) are accumulated in the cell wall of various red algae (Rhodophyta) species. These polysaccharides are of commercial importance in the food, pharmaceutical and biotechnology industries due to their unique physicochemical properties. Although having received significant research attention over the last 20 years, events regarding their biosynthesis have not been elucidated. Aiming for the identification of galactosyltransferase (GalT) genes involved in sulfated galactan biosynthesis, cDNA expression libraries were constructed from the prolific agar-producing South African red seaweed Gelidium pristoides (Turner) Kützing and screened by functional complementation of UDP-galactose 4-epimerase deficient mutants (E. coli and S. cerevisiae). Regretfully, no GalTs were identified. The study however yielded the first UGE enzyme described for a red seaweed. Southern hybridization indicated the presence of two UGE copies and confirmed the gene originated from G. pristoides. Bioinformatic analysis of G. pristoides UGE shows amino acid sequence homology to known UGEs from various organisms. The enzyme was shown to be functional in E. coli crude extracts and showed affinity for UDP-D-galactose, similar to other UDP-galactose 4-epimerases. Further, the isolated G. pristoides UGE (GpUGE) was biochemically characterized and its kinetic parameters determined. We found that there was no kinetic difference between this enzyme and previously described UGE enzymes except enhanced activity in the presence of exogenously added NAD+. The UDP-galactose 4-epimerase (UDP-glucose 4-epimerase, UGE, EC 18.104.22.168) is an essential Leloir pathway enzyme facilitating the catalytic inter-conversion between UDP-D-glucose and UDP-D-galactose. UDP-D-galactose is the nucleotide sugar required by galactosyltransferases for the production of red algae sulfated galactans. UGE is suspected as being responsible for supplying UDP-D-galactose for the synthesis of sulfated galactans. In planta monitoring of GpUGE transcript levels with respect to dark and light cycling indicated high expression of the enzyme at night, while expression diminished during the day. The occurrence of increased nocturnal UGE expression correlates with floridean starch breakdown at night. Evidence for hydrolysis of floridean starch is also reflected in obtained G. pristoides transcriptome sequence data. In red algae, floridean starch degradation coincides with sulfated galactan production. The detection of starch hydrolysis enzyme transcripts alongside increased expression of GpUGE suggests the enzyme plays a role in supplying UDP-Dgalactose for sulfated galactan production. As far as we know, this the first report of sequencing and biochemical characterization of a UGE from red seaweed.