Molecular cloning, characterization, and comparison of UDP-glucose pyrophosphorylase from Gracilaria chouae and Saccharina japonica

Uridine diphosphate (UDP)-glucose pyrophosphorylase (UGPase) is an essential enzyme in algae, catalyzing the phosphorylation that forms UDP-glucose, the key precursor for biosynthesis of carbohydrates such as trehalose, cellulose, and starch. However, the current knowledge about algal UGP is quite little. Here, we conducted transcriptomic and genomic sequence analyses for algal UGPs. Only one UGP sequence was predicted in each red and brown algal species, and a particular UGP/phosphoglucomutase (PGM) fusion gene was found in some brown algae. Sequence comparisons indicated that these predicted UGPs exhibited significant identity to their fungal and plant orthologs, and the fusion UGP/PGM also had active sites conserved in eukaryotes. Phylogenetic analysis suggested that eukaryotic algal and plant UGPs may originate from non-cyanobacterial prokaryotes via horizontal gene transfer, and the non-fusion UGP is the ancestral form. During the evolution of brown algae, UGP had undergone gene duplication and merged with a PGM segment, leading to UGP/PGM. This duplication may not exist in the common ancestor of brown algae and diatoms, but possibly occurred after species differentiation. To study the biochemical properties of algal UGPase, Gracilaria chouae UGP (GcUGP), Saccharina japonica UGP (SjUGP), and S. japonica UGP/PGM (SjUGP/PGM) were recombinantly expressed. GcUGP and SjUGP proteins catalyzed the phosphorylation reaction, with GcUGP having a higher catalytic efficiency (k (cat) /K (m) ) than SjUGP, and the optimal reaction conditions differed partly between these two species, possibly due to their divergent developmental processes and adaptabilities to marine environment. However, little UGPase or PGM activities were detected in fusion protein, which indicates the gene duplication may lead to gene neofunctionalization. The results offer a theoretical basis for further study of the role of UGPase in algae and algal functional product synthesis pathways.