title: Biosynthesis of dTDP-3-acetamido-3,6-dideoxy-alpha-D-glucose.
abstract: Derivatives of 3-amino-3,6-dideoxyhexoses are widespread in Nature. They are part of the repeating units of lipopolysaccharide O-antigens, of the glycan moiety of S-layer (bacterial cell surface layer) glycoproteins and also of many antibiotics. In the present study, we focused on the elucidation of the biosynthesis pathway of dTDP-alpha-D-Quip3NAc (dTDP-3-acetamido-3,6-dideoxy-alpha-D-glucose) from the Gram-positive, anaerobic, thermophilic organism Thermoanaerobacterium thermosaccharolyticum E207-71, which carries Quip3NAc in its S-layer glycan. The biosynthesis of dTDP-alpha-D-Quip3NAc involves five enzymes, namely a transferase, a dehydratase, an isomerase, a transaminase and a transacetylase, and follows a pathway similar to that of dTDP-alpha-D-Fucp3NAc (dTDP-3-acetamido-3,6-dideoxy-alpha-D-galactose) biosynthesis in Aneurinibacillus thermoaerophilus L420-91(T). The ORFs (open reading frames) of interest were cloned, overexpressed in Escherichia coli and purified. To elucidate the enzymatic cascade, the different products were purified by HPLC and characterized by NMR spectroscopy. The initiating reactions catalysed by the glucose-1-phosphate thymidylyltransferase RmlA and the dTDP-D-glucose-4,6-dehydratase RmlB are well established. The subsequent isomerase was shown to be capable of forming a dTDP-3-oxo-6-deoxy-D-glucose intermediate from the RmlB product dTDP-4-oxo-6-deoxy-D-glucose, whereas the isomerase involved in the dTDP-alpha-D-Fucp3NAc pathway synthesizes dTDP-3-oxo-6-deoxy-D-galactose. The subsequent reaction steps of either pathway involve a transaminase and a transacetylase, leading to the specific production of nucleotide-activated 3-acetamido-3,6-dideoxy-alpha-D-glucose and 3-acetamido-3,6-dideoxy-alpha-D-galactose respectively. Sequence comparison of the ORFs responsible for the biosynthesis of dTDP-alpha-D-Quip3NAc revealed homologues in Gram-negative as well as in antibiotic-producing Gram-positive bacteria. There is strong evidence that the elucidated biosynthesis pathway may also be valid for LPS (lipopolysaccharide) O-antigen structures and antibiotic precursors.
authors.affiliation: Zentrum für NanoBiotechnologie, Universität für Bodenkultur Wien, A-1180 Wien, Austria.
authors.forename: Andreas
authors.initials: A
authors.lastname: Pföstl
authors.forename: Sonja
authors.initials: S
authors.lastname: Zayni
authors.forename: Andreas
authors.initials: A
authors.lastname: Hofinger
authors.forename: Paul
authors.initials: P
authors.lastname: Kosma
authors.forename: Christina
authors.initials: C
authors.lastname: Schäffer
authors.forename: Paul
authors.initials: P
authors.lastname: Messner
mesh_terms: D000107:Acetylation; D001483:Base Sequence; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D017931:DNA Primers; D004269:DNA, Bacterial; D003837:Deoxy Sugars / Q000096:biosynthesis / Q000097:blood*; D004798:Enzymes / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D016133:Polymerase Chain Reaction; D013379:Substrate Specificity; D046950:Thermoanaerobacterium / Q000201:enzymology; D013942:Thymine Nucleotides / Q000096:biosynthesis*
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D003837:Deoxy Sugars; D004798:Enzymes; D013942:Thymine Nucleotides; C527679:dTDP-3-acetamido-3,6-dideoxyglucose