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J. Biol. Chem., Vol. 276, Issue 8, 5577-5583, February 23, 2001

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Identification of Two GDP-6-deoxy-D-lyxo-4-hexulose Reductases Synthesizing GDP-D-rhamnose in Aneurinibacillus thermoaerophilus L420-91^T*

Bernd Kneidinger, Michael Graninger, Gerhard Adam^§, Michael Puchberger^¶, Paul Kosma^¶, Sonja Zayni, and Paul Messner

From the  Zentrum für Ultrastrukturforschung und Ludwig Boltzmann-Institut für Molekulare Nanotechnologie, Universität für Bodenkultur Wien, A-1180 Wien, Austria, the ^§ Zentrum für Angewandte Genetik, Universität für Bodenkultur Wien, A-1190 Wien, Austria, and the ^¶ Institut für Chemie, Universität für Bodenkultur Wien, A-1190 Wien, Austria

The glycan repeats of the surface layer glycoprotein of Aneurinibacillus thermoaerophilus L420-91^T 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--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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AF317224.

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