Characterization of Mannuronan C-5-Epimerase Genes from the Brown Alga Laminaria digitata  

Abstract

Alginate is an industrially important polysaccharide obtained commercially by harvesting brown algae. The final step in alginate biosynthesis, the epimerization of ␤-1,4-d-mannuronic acid to ␣-1,4-l-guluronic acid, a structural change that controls the physicochemical properties of the alginate, is catalyzed by the enzyme mannuronan C-5-epimerase. Six different cDNAs with homology to bacterial mannuronan C-5-epimerases were isolated from the brown alga Laminaria digitata (Phaeophyceae). Hydrophobic cluster analysis indicated that the proteins encoded by the L. digitata sequences have important structural similarities to the bacterial mannuronan C-5-epimerases, including conservation of the catalytic site. The expression of the C-5-epimerase genes was examined by northern-blot analysis and reverse transcriptase-polymerase chain reaction in L. digitata throughout a year. Expression was also monitored in protoplast cultures by northern and western blot, reverse transcriptase-polymerase chain reaction, and activity measurements. From both the structural comparisons and the expression pattern, it appears that the cDNAs isolated from L. digitata encode functional mannuronan C-5-epimerases. The phylogenetic relationships of the bacterial and brown algal enzymes and the inferences on the origin of alginate biosynthetic machinery are discussed.Kelps are photosynthetic organisms that are abundant on rocky coasts from temperate to sub-Antarctic and Arctic seas. They are members of the class Phaeophyceae in the division Heterokontophyta, a phylum that emerged as an independent lineage contemporarily with the radiation of other higher eukaryote lineages (Baldauf et al., 2000). They have a haplodiplophasic life cycle, alternating between a microscopic gametophyte phase and a macroscopic sporophyte phase. Although the gametophyte phase consists of uniseriate filaments, a few millimeters long, the sporophytes have distinct organs, i.e. holdfast, stipe, and blade, and can reach from several to 50 m in length depending on the species. The kelp sporophytes are an economically important marine crop, cultivated in East Asia and harvested from natural populations in Europe and North America, including for the production of alginate, a gelforming polysaccharide used in a large variety of pharmaceutical, food, and industrial applications (Onsøyen, 1996).Alginate is the major matrix component of brown algal cell walls, up to 45% in dry weight (Kloareg and Quatrano, 1988). It consists of an unbranched polysaccharide made of ␤-1,4-d-mannuronic acid (M) and its C-5-epimer ␣-1,4-l-guluronic acid (G), which are arranged in homopolymeric regions of M and G blocks, interspaced with regions of alternating structure (MG blocks). These features vary according to the nature and the age of the tissues as well as to the season and to the growing area (sheltered or exposed). The stipe of Laminaria spp., for example, contains a high fraction of G blocks, whereas the blade contains a higher proportion of M residues (Haug et al., 1974). The biological...

Comparative Characterization of Two Marine Alginate Lyases from Zobellia galactanivorans Reveals Distinct Modes of Action and Exquisite Adaptation to Their Natural Substrate

“…These evolutionary constraints likely explain the loss of acetyl groups in algal alginates together with the increased importance of the G units and of their interactions with calcium ions to control gel properties. This hypothesis is consistent with the expansion of the mannuronan C5-epimerase gene families observed in the brown algae Laminaria digitata (17)(18)(19) and E. siliculosus (13).…”

Epimerase Active Domain of Pseudomonas aeruginosa AlgG, a Protein That Contains a Right-Handed β-Helix

“…Finally, model evaluation confirmed that AlgG is likely to have the RH␤H fold. A recent study using hydrophobic cluster analysis showed that an epimerase from the marine brown alga Laminaria digitata has a secondary structure similar to that of P. aeruginosa AlgG (46). Our modeling studies also show a similar fold for the A-module of the extracellular alginate epimerases from A. vinelandii (data not shown).…”

Analysis of G-Block Distributions and Their Impact on Gel Properties of in Vitro Epimerized Mannuronan

“…Even in samples with the highest conversion, the longest homopolymeric G-blocks are still much shorter than what we find in alginate from algae. We have shown earlier 18 of genes coding for epimerases 31 , but no single enzyme has yet been expressed and characterized. It is however established that the G-content increase associated with the age of algal tissue, suggests that long G-blocks are synthesized over an extended period of time.…”