Identification of two GDP-6-deoxy-D-lyxo-4-hexulose reductases synthesizing GDP-D-rhamnose in Aneurinibacillus thermoaerophilus L420-91T.
The glycan repeats of the surface layer glycoprotein of Aneurinibacillus thermoaerophilus L420-91T contain d-rhamnose and 3-acetamido-3,6-dideoxy-d-galactose, both of which are also constituents of lipopolysaccharides of Gram-negative plant and human pathogenic bacteria. The two genes required for biosynthesis of the nucleotide-activated precursor GDP-d-rhamnose, gmd and rmd, were cloned, sequenced, and overexpressed in Escherichia coli. The corresponding enzymes Gmd and Rmd were purified to homogeneity, and functional studies were performed. GDP-d-mannose dehydratase (Gmd) converted GDP-d-mannose to GDP-6-deoxy-d-lyxo-4-hexulose, with NADP+ as cofactor. The reductase Rmd catalyzed the second step in the pathway, namely the reduction of the keto-intermediate to the final product GDP-d-rhamnose using both NADH and NADPH as hydride donor. The elution behavior of the intermediate and end product was analyzed by high performance liquid chromatography. Nuclear magnetic resonance spectroscopy was used to identify the structure of the final product of the reaction sequence as GDP-alpha-d-rhamnose. This is the first characterization of a GDP-6-deoxy-d-lyxo-4-hexulose reductase. In addition, Gmd has been shown to be a bifunctional enzyme with both dehydratase and reductase activities. So far, no enzyme catalyzing these two types of reactions has been identified. Both Gmd and Rmd are members of the SDR (short chain dehydrogenase/reductase) protein family.
Kneidinger
B
B
Zentrum für Ultrastrukturforschung und Ludwig Boltzmann-Institut für Molekulare Nanotechnologie, Universität für Bodenkultur Wien, A-1180 Wien, Austria.
Graninger
M
M
Adam
G
G
Puchberger
M
M
Kosma
P
P
Zayni
S
S
Messner
P
P
D000595:Amino Acid Sequence; D001406:Bacillaceae / Q000201:enzymology*; D001426:Bacterial Proteins / Q000378:metabolism; D003001:Cloning, Molecular; D006023:Glycoproteins / Q000378:metabolism; D006155:Guanosine Diphosphate Mannose / Q000378:metabolism; D006156:Guanosine Diphosphate Sugars / Q000096:biosynthesis*; D006836:Hydro-Lyases / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D009876:Operon; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D050837:Oxidoreductases Acting on Aldehyde or Oxo Group Donors; D011499:Protein Processing, Post-Translational; D012210:Rhamnose / Q000096:biosynthesis; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; C422949:GDP-rhamnose; D006023:Glycoproteins; D006156:Guanosine Diphosphate Sugars; D008565:Membrane Proteins; D006155:Guanosine Diphosphate Mannose; D010088:Oxidoreductases; C115750:GDP-6-deoxy-D-lyxo-4-hexulose reductase; D050837:Oxidoreductases Acting on Aldehyde or Oxo Group Donors; C422948:GDP-D-mannose dehydratase, bacteria; D006836:Hydro-Lyases; D012210:Rhamnose