Biosynthesis of dTDP-3-acetamido-3,6-dideoxy-alpha-D-galactose in Aneurinibacillus thermoaerophilus L420-91T.
The glycan chain of the S-layer protein of Aneurinibacillus thermoaerophilus L420-91T (DSM 10154) consists of d-rhamnose and 3-acetamido-3,6-dideoxy-d-galactose (d-Fucp3NAc). Thymidine diphosphate-activated d-Fucp3NAc serves as precursor for the assembly of structural polysaccharides in Gram-positive and Gram-negative organisms. The biosynthesis of dTDP-3-acetamido-3,6-dideoxy-alpha-d-galactose (dTDP-d-Fucp3NAc) involves five enzymes. The first two steps of the reaction are catalyzed by enzymes that are part of the well studied dTDP-l-rhamnose biosynthetic pathway, namely d-glucose-1-phosphate thymidyltransferase (RmlA) and dTDP-d-glucose-4,6-dehydratase (RmlB). The enzymes catalyzing the last three synthesis reactions have not been characterized biochemically so far. These steps include an isomerase, a transaminase, and a transacetylase. We identified all five genes involved by chromosome walking in the Gram-positive organism A. thermoaerophilus L420-91T and overexpressed the three new enzymes heterologously in Escherichia coli. The activities of these enzymes were monitored by reverse phase high performance liquid chromatography, and the intermediate products formed were characterized by 1H and 13C nuclear magnetic resonance spectroscopy analysis. Alignment of the newly identified proteins with known sequences revealed that the elucidated pathway in this Gram-positive organism may also be valid in the biosynthesis of the O-antigen of lipopolysaccharides of Gram-negative organisms. The key enzyme in the biosynthesis of dTDP-d-Fucp3NAc has been identified as an isomerase, which converts the 4-keto educt into the 3-keto product, with concomitant epimerization at C-4 to produce a 6-deoxy-d-xylo configuration. This is the first report of the functional characterization of the biosynthesis of dTDP-d-Fucp3NAc and description of a novel type of isomerase capable of synthesizing dTDP-6-deoxy-d-xylohex-3-ulose from dTDP-6-deoxy-d-xylohex-4-ulose.
Zentrum für Ultrastrukturforschung und Ludwig Boltzmann-Institut für Molekulare Nanotechnologie, Universität für Bodenkultur Wien, A-1180 Wien, Austria.
Andreas
A
Pfoestl
Andreas
A
Hofinger
Paul
P
Kosma
Paul
P
Messner
D000116:Acetylgalactosamine / Q000031:analogs & derivatives / Q000096:biosynthesis*; D000595:Amino Acid Sequence; D001406:Bacillaceae / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002238:Carbohydrate Epimerases / Q000235:genetics / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D006836:Hydro-Lyases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D005810:Multigene Family; D019906:Nuclear Magnetic Resonance, Biomolecular; D009713:Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D013942:Thymine Nucleotides / Q000096:biosynthesis*
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D013942:Thymine Nucleotides; C476614:dTDP-3-acetamido-3,6-dideoxygalactose; D009713:Nucleotidyltransferases; C020587:glucose-1-phosphate thymidylyltransferase; D006836:Hydro-Lyases; C022771:dTDPglucose 4,6-dehydratase; D002238:Carbohydrate Epimerases; D000116:Acetylgalactosamine