Cloning and functional expression of a novel GDP-6-deoxy-D-talose synthetase from Actinobacillus actinomycetemcomitans.
Actinobacillus actinomycetemcomitans is a Gram-negative coccobacillus that can cause various forms of severe periodontitis and other nonoral infections in human patients. The serotype a-specific polysaccharide antigen of A. actinomycetemcomitans contains solely 6-deoxy-D-talose and its O-2 acetylated modification. This polysaccharide is synthesized from the donor GDP-6-deoxy-D-talose with the relevant talosylation enzyme(s). In the synthesis of GDP-6- deoxy-D-talose, GDP-D-mannose is first converted by GDP-mannose-4,6-dehydratase (GMD) to GDP-4-keto-6-deoxy-D-mannose and then reduced to GDP-6-deoxy-D-talose by GDP-6-deoxy-D-talose synthetase (GTS). In this study, we cloned and overexpressed in Escherichia coli the A. actinomycetemcomitans GTS enzyme responsible for the synthesis of GDP-6-deoxy-D-talose. The recombinant A. actinomycetemcomitans GTS enzyme expressed in E. coli converted the GDP-4-keto-6-deoxy-intermediate to a novel GDP-deoxyhexose. The synthesized GDP-deoxyhexose was shown to be GDP-6-deoxy-D-talose by HPLC, MALDI-TOF MS, and NMR spectroscopy. The functional expression of gts provides another enzymatically defined pathway for the synthesis of GDP-deoxyhexoses, which can be used as donors for the corresponding glycosyltransferases.
Mäki
Minna
M
Department of Bacteriology and Immunology, Haartman Institute and Biomedicum, PO Box 63, FIN-00014 University of Helsinki, Helsinki, Finland.
Järvinen
Nina
N
Räbina
Jarkko
J
Maaheimo
Hannu
H
Mattila
Pirkko
P
Renkonen
Risto
R
D016976:Aggregatibacter actinomycetemcomitans / Q000201:enzymology / Q000235:genetics*; D000595:Amino Acid Sequence; D002238:Carbohydrate Epimerases / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003837:Deoxy Sugars / Q000096:biosynthesis / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D016480:Helicobacter pylori / Q000201:enzymology / Q000235:genetics; D006601:Hexoses / Q000096:biosynthesis / Q000737:chemistry; D006801:Humans; D006836:Hydro-Lyases / Q000235:genetics / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D015394:Molecular Structure; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D016339:Spectrometry, Mass, Fast Atom Bombardment / Q000379:methods
D003837:Deoxy Sugars; D006601:Hexoses; D011994:Recombinant Proteins; C011754:6-deoxytalose; C422948:GDP-D-mannose dehydratase, bacteria; D006836:Hydro-Lyases; D002238:Carbohydrate Epimerases; C480279:GDP-6-deoxy-D-talose synthetase, Actinobacillus actinomycetemcomitans