In Azospirillum brasilense, a gram‐negative nitrogen‐fixing bacterium, l‐arabinose is converted to α‐ketoglutarate through a nonphosphorylative metabolic pathway. In the first step in the pathway, l‐arabinose is oxidized to l‐arabino‐γ‐lactone by NAD(P)‐dependent l‐arabinose 1‐dehydrogenase (AraDH) belonging to the glucose‐fructose oxidoreductase/inositol dehydrogenase/rhizopine catabolism protein (Gfo/Idh/MocA) family. Here, we determined the crystal structures of apo‐ and NADP‐bound AraDH at 1.5 and 2.2 Å resolutions, respectively. A docking model of l‐arabinose and NADP‐bound AraDH and structure‐based mutational analyses suggest that Lys91 or Asp169 serves as a catalytic base and that Glu147, His153, and Asn173 are responsible for substrate recognition. In particular, Asn173 may play a role in the discrimination between l‐arabinose and d‐xylose, the C4 epimer of l‐arabinose.

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