| Summary: | 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 5.1.3.2) 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.
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