Biosynthesis of dTDP-3-acetamido-3,6-dideoxy-{alpha}-D-glucose (dTDP-{alpha}-D-Quip3NAc)

Derivatives of 3amino-3,6-dideoxyhexoses are widely spread in nature. They are part of the repeating units of lipopolysaccharide O-antigens, of the glycan moiety of surface (S-) layer glycoproteins, and also of many antibiotics. In this study, we focused on the elucidation of the biosynthesis pathway of dTDP-3-acetamido-3,6 dideoxy-α-D-glucose (dTDP-α-D-Quip3NAc) from the Gram-positive, anaerobic, thermophilic organism Thermoanaerobacterium thermosaccharolyticum E207-71, which carries Quip3NAc in its S-layer glycan. The biosynthesis of dTDP-α-D-Quip3NAc involves five enzymes - a transferase, a dehydratase, an isomerase, a transaminase, and a transacetylase - and follows a pathway similar to that of dTDP-3-acetamido-3,6-dideoxy-α-D-galactose (dTDP-α-D-Fucp3NAc) biosynthesis in Aneurinibacillus thermoaerophilus L420-91 T}. The open reading frames of interest were cloned, overexpressed in E. coli, and purified. To elucidate the enzymatic cascade, the different products were purified by HPLC and characterized by nuclear magnetic resonance spectroscopy. The initiating reactions catalyzed by the glucose-1-phosphate thymidylyl-transferase 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-keto-6-deoxy-D-glucose intermediate from the RmlB product dTDP-4-keto-6-deoxy-D-glucose, whereas the isomerase involved in the dTDP-α-D-Fucp3NAc pathway synthesizes dTDP-3-keto-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-α-D-glucose and 3-acetamido-3,6-dideoxy-α-D-galactose, respectively. Sequence comparison of the open reading frames responsible for the biosynthesis of dTDP-α-D-Quip3NAc revealed homologs 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 O-antigen structures and antibiotic precursors.