Identification of two GDP-6-deoxy-D-lyxo-4-hexulose reductases synthesizing GDP-D-rhamnose in Aneurinibacillus thermoaerophilus L420-91(T)

The glycan repeats of the surface layer glycoprotein of Aneurinibacillus th ermoaerophilus L420-91(T) contain D-rhamnose and 3-acetamido-3,6-dideoxy-D- galactose, both of which are also constituents of lipopolysaccharides of Gr am-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 co rresponding enzymes Gmd and Rmd were purified to homogeneity, and functiona l studies were performed. GDP-D-mannose dehydratase (Gmd) converted GDP-D-m annose to GDP-6-deoxy-D-lyxo-4-hexulose, with NADP(+) as cofactor. The redu ctase 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 en d product was analyzed by high performance liquid chromatography. Nuclear m agnetic resonance spectroscopy was used to identify the structure of the fi nal product of the reaction sequence as GDP-cr-D-rhamnose. This is the firs t characterization of a GDP-6-deoxy-D-lyxo-4-hexulose reductase. In additio n, Gmd has been shown to be a bifunctional enzyme with both dehydratase and reductase activities. So far, no enzyme catalyzing these two types of reac tions has been identified. Both Gmd and Rmd are members of the SDR (short c hain dehydrogenase/reductase) protein family.