Rhamnose synthase (RHM) is a key enzyme in the biosynthesis of uridine diphosphate rhamnose (UDP-Rha), reversibly converting uridine diphosphate-glucose (UDP-Glc) into UDP-Rha in the presence of NADH or NADPH. In this research, yeast extract (YE) was used to stimulate Sorbus aucuparia suspension cells. Based on a previous study of the transcriptome database of S. aucuparia suspension cells, two RHMs were cloned from S. aucuparia and named SaRHM1 (GenBank No.: MK213340) and SaRHM2 (GenBank No.: MK213341). The SaRHM1 gene contained a 2 007 bpopen reading frame (ORF) encoding a polypeptide of 668 amino acids with a molecular weight of 75.25 kD, and a theoretical isoelectric point (pI) of 7.24. The SaRHM2 gene contained a 2 040 bpORF encoding a polypeptide of 679 amino acids with a molecular weight of 76.26 kD and pI of 6.41. Bioinformatic analysis indicated that SaRHM1 and SaRHM2 contained two special sequences of GxxGxxG/A and YxxxK. Multiple sequence alignments and phylogenetic trees show that SaRHM1 and SaRHM2 have high sequence similarity with other plant species of RHMs. The results of enzyme activity assays in vitro revealed that both recombinant SaRHM1 and SaRHM2 are able to convert UDP-Glc into UDP-Rha. SaRHMs displayed maximum activity at 40 ℃ and a pH of 8 and 9, respectively. The Km values of SaRHM1 and SaRHM2 for UDP-Glc were 212.4 ± 56.70 and 361.0 ± 63.74 μmol·L-1, respectively, with Vmax values of 235.5 ± 18.98 and 516.5 ± 22.30 nmol·min-1·μg-1, respectively. This study reports the cloning and sequencing of RHMs from S. aucuparia and verifies their function, which likely provide rhamnose donors for the subsequent biosynthesis of rhamnosides.
| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |