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crystal structures of ManD and its complex with D-mannonate is solved. It shows that the insert sequence forms two alpha helices locating above the active site. The two insert alpha helices introduce a loop that forms a cap covering the substrate binding pocket, which restricts the tunnels of substrate entering and product releasing from the active site
hanging drop method at room temperature. Structures of the wild type ManD from are determined at pH 7.5 in the presence of Mg2+ and also in the presence of Mg2+ and the 2-keto-3-keto-D-gluconate dehydration product. The structure of the catalytically active K271E mutant is determined at pH 5.5 in the presence of the D-mannonate substrate
ManD in native form and in complex with its substrate D-mannonate and Mn2+ ion, hanging drop vapor diffusion method, using 0.2 M potassium-sodium tartrate, 0.1 M sodium citrate (pH 6.5), and 1 M ammonium sulfate (native form) or using 0.2 M potassium sodium tartrate tetrahydrate, 0.1 M trisodium citrate dehydrate (pH 6.5), 1 M ammonium sulfate, and 15 mM D-mannonate