Functional analyses of OcRhS1 and OcUER1 involved in UDP-L-rhamnose biosynthesis in Ornithogalum caudatum.
UDP-L-rhamnose (UDP-Rha) is an important sugar donor for the synthesis of rhamnose-containing compounds in plants. However, only a few enzymes and their encoding genes involved in UDP-Rha biosynthesis are available in plants. Here, two genes encoding rhamnose synthase (RhS) and bi-functional UDP-4-keto-6-deoxy-D-glucose (UDP-4K6DG) 3, 5-epimerase/UDP-4-keto-L-rhamnose (UDP-4KR) 4-keto-reductase (UER) were isolated from Ornithogalum caudatum based on the RNA-Seq data. The OcRhS1 gene has an ORF (open reading frame) of 2019 bp encoding a tri-functional RhS enzyme. In vitro enzymatic assays revealed OcRhS1 can really convert UDP-D-glucose (UDP-Glc) into UDP-Rha via three consecutive reactions. Biochemical evidences indicated that the recombinant OcRhS1 was active in the pH range of 5-11 and over the temperature range of 0-60 °C. The Kmvalue of OcRhS1 for UDP-Glc was determined to be 1.52 × 10-4M. OcRhS1 is a multi-domain protein with two sets of cofactor-binding motifs. The cofactors dependent properties of OcRhS1 were thus characterized in this research. Moreover, the N-terminal portion of OcRhS1 (OcRhS1-N) was observed to metabolize UDP-Glc to form intermediate UDP-4K6DG. OcUER1 contains an ORF of 906 bp encoding a polypeptide of 301 aa. OcUER1 shared high similarity with the carboxy-terminal domain of OcRhS1 (OcRhS1-C), suggesting its intrinsic ability of converting UDP-4K6DG into UDP-Rha. It was thus reasonably inferred that UDP-Glc could be bio-transformed into UDP-Rha under the collaborating action of OcRhS1-N and OcUER1. The subsequently biochemical assay verified this notion. Importantly, expression profiles of OcRhS1 and OcUER1 revealed their possible involvement in the biosynthesis of rhamnose-containing polysaccharides in O. caudatum.
Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College (State Key Laboratory of Bioactive Substance and Function of Natural Medicines & Ministry of Health Key Laboratory of Biosynthesis of Natural Products), Beijing, 100050, China.
Sen
S
Yin
Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College (State Key Laboratory of Bioactive Substance and Function of Natural Medicines & Ministry of Health Key Laboratory of Biosynthesis of Natural Products), Beijing, 100050, China.
Ming
M
Liu
Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College (State Key Laboratory of Bioactive Substance and Function of Natural Medicines & Ministry of Health Key Laboratory of Biosynthesis of Natural Products), Beijing, 100050, China. Electronic address: jianqiangk@imm.ac.cn.
Jian-Qiang
JQ
Kong
D000595:Amino Acid Sequence; D053898:Biosynthetic Pathways; D002237:Carbohydrate Dehydrogenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002238:Carbohydrate Epimerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017343:Genes, Plant; D007700:Kinetics; D031442:Ornithogalum / Q000235:genetics* / Q000378:metabolism*; D010802:Phylogeny; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012210:Rhamnose / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014539:Uridine Diphosphate Sugars / Q000096:biosynthesis* / Q000235:genetics
D010940:Plant Proteins; D011994:Recombinant Proteins; D014539:Uridine Diphosphate Sugars; D002237:Carbohydrate Dehydrogenases; D002238:Carbohydrate Epimerases; D012210:Rhamnose
5-epimerase/UDP-4-keto-L-rhamnose 4-keto-reductase; Anti-cancer polysaccharides; O. caudatum; Rhamnose synthase; UDP-4-keto-6-deoxy-D-glucose 3; UDP-L-rhamnose