Study On GDP-mannose Dehydrogenase Genes And Phomannomutase Genes In Saccharina Japonica

GDP-mannose dehydrogenase(GMD) is the rate-limiting enzyme which involved in the synthesis of alginate, but no GMD gene from S. japonica has been characterized so far. In this study, two GMD genes from S. japonica(Sjgmd1, Sjgmd2), which belonged to GDP-mannose dehydrogenase/UDP-glucose dehydrogenase family, were cloned by RACE method. The open reading frame(ORF) length of Sjgmd1 and Sjgmd2 is 963 bp and 948 bp, respectively, which showed 70.06% identity. Random coil and α-helix were the common major secondary structure for Sjgmd1 and Sjgmd2. The multiple sequences alignment and three-dimensional structures analysis proposed a novel binding and catalysis mechnaism in SjGMD. The fusion protein(SjGMD) were expressed successfully and the enzyme assays results showed the optimal temperature were 30℃(SjGMD1) and 20℃(SjGMD2), and optimal pH value were 8.0(SjGMD1) and 8.25(SjGMD2). The Km for GDP-mannose were 289 μM(SjGMD1) and 177 μM(SjGMD2), respectively.In addition, two phosphomannomutase(PMM) genes of S. japonica(Sjpmm1、Sjpmm2), which involved in the synthesis of alginate and fucoidan, were cloned by RACE method. The homologous analysis indicated that Sjpmm1 belonged to the HAD superfamily, and the ORF length was 759 bp, encoding 252 amino acids with the molecular weight(MV) of 28.51 kDa; while Sjpmm2 belonged to the phosphohexomutase superfamily, and the ORF length was 1866 bp, encoding 621 amino acids with the MV of 66.49 kDa. The α-helix was regarded as the commom major secondary structure to SjPMM1 and SjPMM2. The phylogenetic analysis showed that Sjpmm1 was evolved from the ancient eukaryotes, while Sjpmm2 was from the bacteria by horizontal gene transfer. The qRT-PCR analysis indicated that temperature could increase the transcription of Sjpmm, which may cause the more synthesized of fucoidan. Furthermore, high-concentration fusion protein of SjPMM1 was expressed, which could be applied for further function analysis.