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D-rhamnose is a rare 6-deoxy monosaccharide primarily found in the lipopolysaccharide of pathogenic bacteria, where it is involved in host-bacterium interactions and the establishment of infection. The biosynthetic pathway of D-rhamnose proceeds through two steps. GDP-D-mannose 4,6-dehydratase (GMD) first converts GDP-D-mannose to the 4-keto-6-deoxy intermediate, which is subsequently reduced to the nucleotide-activated D-rhamnose by a reductase. We have determined the crystal structure of a bacterial GMD in complex with NADPH and GDP. GMD belongs to the nucleotide-sugar modifying subfamily of the short-chain dehydrogenase/reductase (SDR) enzymes. SDRs are known to have homologous tertiary structures and share a conserved catalytic triad of Tyr-xxx-Lys and Ser/Thr. GMD has a bi-domain structure consistent with other related members of this subfamily. The larger N-terminal domain, the site of NADPH cofactor binding, consists of a Rossmann fold, a motif commonly associated with dinucleotide binding. The smaller C-terminal domain is responsible for substrate binding. GMD deviates from the typical homodimeric structures seen in most other related enzymes of this subfamily, as it is a tetramer like its plant homolog MUR1, a GDP-D-mannose 4,6-dehydratase isoform from Arabidopsis thaliana¹. At the tetramer interface, the cofactor binding sites are adjoined such that the adenosyl phosphate moieties of the bound NADPH molecules are within 8 Å. A short peptide segment forms a loop in this region that stretches into the neighboring monomer making not only protein-protein interactions, but also hydrogen bonding interactions with the neighboring cofactor. In addition, tetrameric contacts outside of this region are conserved across both the prokaryotic and eukaryotic GMDs. When the residues involved are not conserved, substitutions are conservative and expected to be compatible with tetramer formation. These observations suggest that the tetramer may be a more common oligomeric state for GMDs than previously thought.