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

Abstract

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-α-D-rhamnose.