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© 2021 American Chemical Society.d–Glucosaminate–6–phosphate ammonia–lyase (DGL) is a pyridoxal 5′–phosphate (PLP)–dependent enzyme that produces 2–keto–3–deoxygluconate 6–phosphate (KDG–6–P) in the metabolism of d–glucosaminic acid by Salmonella enterica serovar typhimurium. We have determined the crystal structure of DGL by SAD phasing with selenomethionine to a resolution of 2.58 Ã…. The sequence has very low identity with most other members of the aminotransferase (AT) superfamily. The structure forms an octameric assembly as a tetramer of dimers that has not been observed previously in the AT superfamily. PLP is covalently bound as a Schiff base to Lys–213 in the catalytic dimer at the interface of two monomers. The structure lacks the conserved arginine that binds the α–carboxylate of the substrate in most members of the AT superfamily. However, there is a cluster of arginines in the small domain that likely serves as a binding site for the phosphate of the substrate. The deamination reaction performed in D2O gives a KDG–6–P product stereospecifically deuterated at C3; thus, the mechanism must involve an enamine intermediate that is protonated by the enzyme before product release. Nuclear magnetic resonance (NMR) analysis demonstrates that the deuterium is located in the pro–R position in the product, showing that the elimination of water takes place with inversion of configuration at C3, which is unprecedented for a PLP–dependent dehydratase/deaminase. On the basis of the crystal structure and the NMR data, a reaction mechanism for DGL is proposed.
