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Gewählte Publikation:

Pfoestl, A; Hofinger, A; Kosma, P; Messner, P.
(2003): Biosynthesis of dTDP-3-acetamido-3,6-dideoxy-alpha-D-galactose in Aneurinibacillus thermoaerophilus L420-91T.
J Biol Chem. 2003; 278(29):26410-26417 FullText FullText_BOKU

The glycan chain of the S-layer protein of Aneurinibacillus thermoaerophilus L420-91(T) (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 H-1 and C-13 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.
Autor*innen der BOKU Wien:
Hofinger-Horvath Andreas
Kosma Paul
Messner Paul
Find related publications in this database (using NML MeSH Indexing)
Acetylgalactosamine - analogs & derivatives
Amino Acid Sequence -
Bacillaceae - enzymology
Base Sequence -
Carbohydrate Epimerases - genetics
DNA, Bacterial - genetics
Genes, Bacterial -
Hydro-Lyases - genetics
Molecular Sequence Data -
Multigene Family -
Nuclear Magnetic Resonance, Biomolecular -
Nucleotidyltransferases - genetics
Recombinant Proteins - genetics
Restriction Mapping -
Sequence Homology, Amino Acid -
Thymine Nucleotides - biosynthesis

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