Comparative characterization of two GDP-mannose dehydrogenase genes from Saccharina japonica (Laminariales, Phaeophyceae).

BACKGROUND

Saccharina japonica is an important commercial brown seaweed, its main product is alginate, which is used in food, textile and by the cosmetic and pharmaceutical industries. GDP-mannose dehydrogenase (GMD) is the key enzyme involved in the synthesis of alginate. However, little is known about GMD in S. japonica. Here we report comparative biochemical analysis of two GMD genes in S. japonica.

RESULTS

Two GMD genes from S. japonica (Sjgmd1, Sjgmd2) were cloned. The open reading frame lengths of Sjgmd1, Sjgmd2 are 963 bp and 948 bp, respectively. Alignment analysis showed that the two SjGMD sequences shared 79.38 % identity. Both proteins possess the GGxCLPKDV and GxGxVG sequence motifs characteristic of the short-chain dehydrogenase/reductase superfamily. The optimum temperatures for SjGMDs were 30 °C (SjGMD1) and 20 °C (SjGMD2), and the optimum pH values were 8.0 (SjGMD1) and 8.25 (SjGMD2). Kinetic analysis demonstrated the Km values for the substrate GDP-mannose were 289 μM (SjGMD1) and 177 μM (SjGMD2), and the Km values for the cofactor NAD(+) were 139 μM (SjGMD1) and 195 μM (SjGMD2). The metal iron Zn(2+) is a potent inhibitor of SjGMD1 and SjGMD2. Real-time PCR analysis showed that heat and desiccation treatments resulted in a significant increase in Sjgmd1 and Sjgmd2 transcript abundance, suggesting that the SjGMDs are directly involved in the acclimitisation of S. japonica to abiotic stresses.

CONCLUSION

Our work identified two novel genes encoding GMD in S. japonica, comparatively characterized their structural characteristics and enzyme kinetics, and revealed the function of GMD in the stress adaptability of S. japonica. The knowledge obtained here enriched our understanding of the alginate synthesis mechanism in S. japonica, and may promote further research on functional differences between GMD genes.