Cloning and expression of Helicobacter pylori GDP-l-fucose synthesizing enzymes (GMD and GMER) in Saccharomyces cerevisiae.
Helicobacter pylori is a Gram-negative gastric pathogen causing diseases from mild gastric infections to gastric cancer. The difference in clinical outcome has been suggested to be due to strain differences. H. pylori undergoes phase variation by changing its lipopolysaccharide structure according to the environmental conditions. The O-antigen of H. pylori contains fucosylated glycans, similar to Lewis structures found in human gastric epithelium. These Lewis glycans of H. pylori have been suggested to play a role in pathogenesis in the adhesion of the bacterium to gastric epithelium. In the synthesis of fucosylated structures, GDP-l-fucose is needed as a fucose donor. Here, we cloned the two key enzymes of GDP-l-fucose synthesis, H. pylori gmd coding for GDP-d-mannose dehydratase (GMD), and gmer coding for GDP-4-keto-6-deoxy-d-mannose-3,5-epimerase/4-reductase (GMER) and expressed them in an enzymatically active form in Saccharomyces cerevisiae. The end product of these enzymes, GDP-l-fucose was used as a fucose donor in a fucosyltransferase assay converting sialyl-N-acetyllactosamine to sialyl Lewis X.
Järvinen
N
N
Department of Bacteriology and Immunology, Haartman Institute and Biomedicum, University of Helsinki, Finland.
Mäki
M
M
Räbinä
J
J
Roos
C
C
Mattila
P
P
Renkonen
R
R
D001483:Base Sequence; D015153:Blotting, Western; D002238:Carbohydrate Epimerases / Q000235:genetics* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D017931:DNA Primers; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D005643:Fucose / Q000031:analogs & derivatives* / Q000096:biosynthesis*; D016480:Helicobacter pylori / Q000201:enzymology*; D006836:Hydro-Lyases / Q000235:genetics* / Q000378:metabolism; D007658:Ketone Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D009097:Multienzyme Complexes / Q000235:genetics* / Q000378:metabolism; D009711:Nucleotides / Q000096:biosynthesis*; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D017931:DNA Primers; D029968:Escherichia coli Proteins; C418804:GDP-2F-fucose; D009097:Multienzyme Complexes; D009711:Nucleotides; C110682:wcaG protein, E coli; D005643:Fucose; D007658:Ketone Oxidoreductases; C422948:GDP-D-mannose dehydratase, bacteria; D006836:Hydro-Lyases; D002238:Carbohydrate Epimerases