Mannuronan C5-epimerase Research Articles

Enzymatic characterization of a mannuronan C5-epimerase from the subtropical brown alga Cladosiphon okamuranus

Enzymatic characterization of a mannuronan C5-epimerase from the subtropical brown alga Cladosiphon okamuranus

The Cell Wall Polysaccharides Biosynthesis in Seaweeds: A Molecular Perspective.

Cell wall polysaccharides (CWPS) of seaweeds play crucial roles in mechanical shear resistance, cell-cell adhesion and the interactions with changeable marine environments. They have diverse applications in food, cosmetics, agriculture, pharmaceuticals and therapeutics. The recent boost of multi-omics sequence analysis has rapidly progressed the mining of presumed genes encoding enzymes involved in CWPS biosynthesis pathways. In this review, we summarize the biosynthetic pathways of alginate, fucoidan, agar, carrageenan and ulvan in seaweeds referred to the literatures on published genomes and biochemical characterization of encoded enzymes. Some transcriptomic data were briefly reported to discuss the correlation between gene expression levels and CWPS contents. Mannuronan C-5 epimerase (MC5E) and carbohydrate sulfotransferase (CST) are crucial enzymes for alginate and sulfated CWPS, respectively. Nonetheless, most CWPS-relevant genes were merely investigated by gene mining and phylogenetic analysis. We offer an integrative view of CWPS biosynthesis from a molecular perspective and discuss about the underlying regulation mechanism. However, a clear understanding of the relationship between chemical structure and bioactivities of CWPS is limited, and reverse genetic manipulation and effective gene editing tools need to be developed in future.

Genome-wide transcriptome profiling and characterization of mannuronan C5-epimerases in Saccharina japonica

Alginate is a linear polysaccharide comprised of 1,4-linked-β-D-mannuronic acid (M) and α-L-guluronic acid (G). As the major cell wall polysaccharide in brown algae, alginate is widely used in the food, pharmaceutical, and chemical industries. Mannuronan C5-epimerases (MC5Es) catalyze the specific conversion of M to its C-5 epimer G, playing a key role in the modification of alginate. In this study, 143 MC5E gene sequences from Saccharina japonica (SjMC5Es) were identified based on the genome and transcriptome data. Transcriptomics analysis was conducted, and the results showed that the SjMC5Es varied in their spatial and temporal expression patterns. Most SjMC5Es exhibited higher expression in the basal blades of the algae at earlier developmental stages than at later adult stages, and the transcription levels of the SjMC5Es showed a clear trend of gradual increase or decrease from the basal to the distal blades. In addition, the β-D-mannuronic acid/α-L-guluronic acid (M/G) ratio of alginate from all samples was measured, and the M/G ratios were associated with the transcription levels of the SjMC5E genes. Finally, the highly expressed SjMC5E2 was characterized, soluble SjMC5E2 was heterologously expressed, and its mannuronan C5-epimerase activity was confirmed by 1H-nuclear magnetic resonance analysis. Our results provide a global view of the SjMC5Es in S. japonica and lay a foundation for further applications of MC5Es in improving the physiochemical properties of alginate.

Changes in Cell Wall Structure During Rhizoid Formation of Silvetia babingtonii (Fucales, Phaeophyceae) Zygotes.

We examined the ultrastructure of the cell wall and immunolocalization of alginates using specific antibodies against M-rich alginates and MG blocks during rhizoid formation in fucoid zygotes, Silvetia babingtonii. The thallus region of 24-h-old zygotes had a cell wall made of three layers with different fiber distribution. In the 12-h-old zygotes, three layers in the thallus were observed before rhizoid formation, namely the inner, middle, and outer layers. During rhizoid elongation, only the inner layer was apparent close to the rhizoid tip area. Immunoelectron microscopy detected M-rich blocks of alginate on the inner half of the cell wall, irrespective of the number of layers in the thallus and rhizoid regions. The MG blocks were seen to cover a slightly wider area than M-rich alginate blocks. It was suggested that parts of M in mannuronan would be rapidly converted to G, and MG-blocks are generated. Transcriptome analysis was performed using 3 -, 10 -, and 24-h-old zygotes after fertilization to examine the relationship between gene expression and alginate synthesis over time. The expression of two mannuronan C5-epimerase homologs that convert mannuronic acid into guluronic acid in alginates was upregulated or downregulated over the course of the examination.

Functional heterologous expression and characterization of mannuronan C5-epimerase from the brown alga Saccharina japonica

Brown algae produce alginate that has various ratios and diverse sequences of two uronic acids, β-d-mannuronic acid and α-l-guluronic acid, compared with those of alginate produced by bacteria. This diversity of alginate in brown algae is caused by mannuronan C5-epimerases (MC5Es), which catalyze the conversion of β-d-mannuronic acid to α-l-guluronic acid. Although several bacterial MC5E enzymes have been well characterized, to date, there exists no information on the biochemical properties of eukaryotic MC5E. In this study, MC5E expression was detected in a brown alga Saccharina japonica sporophyte by immunoblot analysis. We also searched for MC5E mRNA from S. japonica by RT-PCR and revealed eight partial amino acid sequences, SjC5-I to -VIII. We focused on the highest frequency clone, SjC5-VI, and elucidated its full-length cDNA and putative gene structure. The translated SjC5-VI protein consists of 499 amino acids, with the N-terminal 21 amino acids predicted as a secretion signal sequence. Functional recombinant SjC5-VI (rSjC5-VI) was successfully expressed as a secreted protein using an insect-cell expression system, and we determined the optimal temperature, pH, and NaCl concentrations to be 35°C, 7.0–8.2, and 300mM, respectively, using the Ca2+-induced gel-formation assay. In addition, Ca2+ enhanced gelation by 1.7-fold following rSjC5-VI activity. Furthermore, 1H-NMR spectroscopy of rSjC5-VI-treated polyM revealed alternate epimerization of β-d-mannuronic acid to α-l-guluronic acid. To the best of our knowledge, this is the first report on the characterization of MC5E activity in eukaryotes.

EXPRESSION PROFILING OF THE MANNURONAN C5-EPIMERASE MULTIGENIC FAMILY IN THE BROWN ALGA LAMINARIA DIGITATA (PHAEOPHYCEAE) UNDER BIOTIC STRESS CONDITIONS(1).

The aim of this study was to profile expression of mannuronan C5-epimerase (ManC5-E) genes of Laminaria digitata (Huds.) J. V. Lamour. under treatment with brown alga cell wall components known to induce defense reaction in this organism. After determining that incubation in the presence of homo-guluronates increased the accumulation of ManC5-E transcripts, we cloned a number of partial ManC5-E genes from nonelicited and elicited specimens. In addition to these fragments, previously identified genes from sporophyte and protoplast cDNA libraries were considered for a clustering analysis. Several groups of ManC5-E genes were observed, including groups containing sequences specific from nonelicited and elicited sporophytes, and sequences isolated from protoplasts. Quantitative real-time PCR experiments were then carried out using genes representative of different groups. Results of transcript accumulation during protoplast regeneration and sporophyte elicitation supported the hypothesis that specific C5-epimerase genes are recruited under pathogen attack, enabling L.digitata to modify its cell wall in response to environmental stimuli.

1H, 15N, 13C resonance assignment of the AlgE6R1 subunit from the Azotobacter vinelandii mannuronan C5-epimerase

Isotopically labelled, (13)C/(15)N from of recombinant subunit of the first R-module from alginate C5-epimerase 6 (AlgE6R1) from Azotobacter vinelandii mannuronan C5-epimerase was produced. We report here the (1)H, (15)N, (13)C resonance assignment of this subunit from AlgE6 epimerase.

Enzymatic modification of alginates with the mannuronan C-5epimerase AlgE4 enhances their solubility at low pH

The recent availability of mannuronan C-5 epimerase from Azotobacter vinelandii opens up for modifying the sequential structure of alginates in a controlled way. To investigate the effect of an increased amount of alternating sequences on the acid solubility of alginate, different alginates from Durvillea antarctica, Lessonia nigrescens, Laminaria hyperborea and a bacterial mannuronan were epimerized using AlgE4. This enzyme is produced recombinantly in Escherichia coli and converts the M blocks into MGM sequences leaving the G-blocks intact. The solubility of the modified alginates was investigated as a function of pH and time.

Effects of molecular weight and elastic segment flexibility on syneresis in Ca-alginate gels

Syneresis in Ca-alginate gels was studied as a function of the alginate molecular weight and the degree of flexibility of the elastic segments. Small angle X-ray scattering of alginate gels reveals an increased lateral association of junction zones when entering a Ca 2+ regime giving syneresis. This suggests growth of junction zones to be the primary driving force for syneresis. Ca-alginate gels prepared from alginates with different molecular weights show a reduced syneresis with decreasing M w. A reduced syneresis is also observed when fractions of a high M w alginate is replaced by short alginate molecules enriched in guluronate residues. The effect of altering the monomer sequence of the elastic segments spanning the junction zones was studied by converting poly-mannuronate regions to alternating guluronate/mannuronate sequences by the mannuronan C5-epimerase AlgE4. This epimerisation reaction gives a more flexible elastic segment. The epimerased alginates yielded gels with larger syneresis compared to the non-epimerased, native alginate samples. Thus, both molecular weight and elastic segment flexibility are needed in a molecular model for describing syneretic behaviour in alginate gels. These parameters will to a large extent determine to which degree the non-equilibrium nature of the alginate gel is macroscopically expressed (syneresis).

A coupled assay for the detection of mannuronan C5 epimerase

A coupled assay for the detection of mannuronan C5 epimerase

Ionic and acid gel formation of epimerised alginates; the effect of AlgE4

AlgE4 is a mannuronan C5 epimerase converting homopolymeric sequences of mannuronate residues in alginates into mannuronate/guluronate alternating sequences. Treating alginates of different biological origin with AlgE4 resulted in different amounts of alternating sequences. Both ionically cross-linked alginate gels as well as alginic acid gels were prepared from the epimerised alginates. Gelling kinetics and gel equilibrium properties were recorded and compared to results obtained with the original non-epimerised alginates. An observed reduced elasticity of the alginic acid gels following epimerisation by AlgE4 seems to be explained by the generally increased acid solubility of the alternating sequences. Ionically (Ca 2+) cross-linked gels made from epimerised alginates expressed a higher degree of syneresis compared to the native samples. An increase in the modulus of elasticity was observed in calcium saturated (diffusion set) gels whereas calcium limited, internally set alginate gels showed no change in elasticity. An increase in the sol–gel transitional rate of gels made from epimerised alginates was also observed. These results suggest an increased possibility of creating new junction zones in the epimerised alginate gel due to the increased mobility in the alginate chain segments caused by the less extended alternating sequences.

Pseudomonas aeruginosa AlgG is a polymer level alginate C5-mannuronan epimerase

Alginate is a viscous extracellular polymer produced by mucoid strains of Pseudomonas aeruginosa that cause chronic pulmonary infections in patients with cystic fibrosis. Alginate is polymerized from GDP-mannuronate to a linear polymer of beta-1-4-linked residues of D-mannuronate and its C5-epimer, L-guluronate. We previously identified a gene called algG in the alginate biosynthetic operon that is required for incorporation of L-guluronate residues into alginate. In this study, we tested the hypothesis that the product of algG is a C5-epimerase that directly converts D-mannuronate to L-guluronate. The DNA sequence of algG was determined, and an open reading frame encoding a protein (AlgG) of approximately 60 kDa was identified. The inferred amino terminus of AlgG protein contained a putative signal sequence of 35 amino acids. Expression of algG in Escherichia coli demonstrated both 60-kDa pre-AlgG and 55-kDa mature AlgG proteins, the latter of which was localized to the periplasm. An N-terminal analysis of AlgG showed that the signal sequence was removed in the mature form. Pulse-chase experiments in both E. coli and P. aeruginosa provided evidence for conversion of the 60- to the 55-kDa size in vivo. Expression of algG from a plasmid inan algG (i.e., polymannuronate-producing) mutant of P. aeruginosa restored production of an alginate containing L-guluronate residues. The observation that AlgG is apparently processed and exported from the cytoplasm suggested that it may act as a polymer-level mannuronan C5-epimerase. An in vitro assay for mannuronan C5 epimerization was developed wherein extracts of E. coli expressing high levels of AlgG were incubated with polymannuronate. Epimerization of D-mannuronate to L-guluronate residues in the polymer was detected enzymatically, using a L-guluronate-specific alginate lyase of Klebsiella aerogenes. Epimerization was also detected in the in vitro reaction between recombinant AlgG and poly-D-mannuronate, using high-performance anion-exchange chromatography. The epimerization reaction was detected only when acetyl groups were removed from the poly-D-mannuronate substrate, suggesting that AlgG epimerization activity in vivo may be sensitive to acetylation of the D-mannuronan residues. These results demonstrate that AlgG has polymer-level mannuronan C5-epimerase activity.