Interaction of GDP-4-keto-6-deoxymannose-3,5-epimerase-4-reductase with GDP-mannose-4,6-dehydratase stabilizes the enzyme activity for formation of GDP-fucose from GDP-mannose.
We cloned the GDP-4-keto-6-deoxymannose-3,5-epimerase-4-reductase gene from Arabidopsis thaliana (AtFX/GER1). The yeast Saccharomyces cerevisiae was transfected with the AtFX/GER1 gene coexpressed with GDP-mannose-4,6-dehydratase gene of A. thaliana (MUR1). In vitro GDP-fucose synthesis activity was observed in the cytoplasmic fraction of cells coexpressing the AtFX/GER1 gene and MUR1 gene. However, the cytoplasmic fraction of cells expressing MUR1 alone did not show the GDP-mannose-4,6-dehydratase activity. This result suggests that the AtFX/GER1 protein may contribute to maintenance of the MUR1 protein as the active form. Immunoprecipitation experiments showed that both proteins interact with each other, indicating that this interaction is required to maintain MUR1 protein as the active or stable form. Finally, in vivo GDP-fucose synthesis activity was analyzed by measuring the amount of GDP-fucose produced in the cytoplasm of yeast cells. The amount of GDP-fucose in cells coexpressing MUR1 and AtFX/GER1 genes was 3.5 times higher than the amount of GDP-mannose in the same cells, indicating that this coexpression system is suitable for production of the valuable sugar nucleotide GDP-fucose in yeast.
Nakayama
Ken-ichi
K
Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology, Tsukuba Central 6, 1-1 Higashi, Tsukuba 305-8566, Japan.
Maeda
Yumi
Y
Jigami
Yoshifumi
Y
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics; D002238:Carbohydrate Epimerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004795:Enzyme Stability; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006154:Guanosine Diphosphate Fucose / Q000096:biosynthesis*; D006155:Guanosine Diphosphate Mannose / Q000096:biosynthesis*; D006836:Hydro-Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007658:Ketone Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011233:Precipitin Tests; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; C110682:wcaG protein, E coli; D006154:Guanosine Diphosphate Fucose; D006155:Guanosine Diphosphate Mannose; D007658:Ketone Oxidoreductases; D006836:Hydro-Lyases; C044971:GDPmannose 4,6-dehydratase; D002238:Carbohydrate Epimerases