RESUMO
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 Å. 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.
Assuntos
Amônia-Liases/metabolismo , Glucosamina/análogos & derivados , Glucose-6-Fosfato/análogos & derivados , Fosfato de Piridoxal/metabolismo , Aminoácidos/metabolismo , Sítios de Ligação , Catálise , Cristalografia por Raios X/métodos , Glucosamina/metabolismo , Glucose-6-Fosfato/metabolismo , Cinética , Liases/metabolismo , Modelos Moleculares , Fosfatos , Bases de Schiff , Especificidade por Substrato , Transaminases/metabolismoRESUMO
Salmonella enterica serovar Typhimurium utilizes a wide range of growth substrates, some of which are relatively novel. One of these unusual substrates is d-glucosaminate, which is metabolized by the enzymes encoded in the dga operon. d-Glucosaminate is transported and converted to d-glucosaminate-6-phosphate (G6P) by a phosphotransferase system, composed of DgaABCD. The protein product of dgaE, d-glucosaminate-6-phosphate ammonia lyase (DGL), converts G6P to 2-keto-3-deoxygluconate-6-phosphate, which undergoes a retroaldol reaction catalyzed by the DgaF protein to give d-glyceraldehyde-3-phosphate and pyruvate. We have now developed an improved synthesis of G6P which gives a higher yield. The DGL reaction is of mechanistic interest because it is one of only a few enzymes in the pyridoxal-5'-phosphate (PLP) dependent aminotransferase superfamily known to catalyze reaction of a d-amino acid substrate. The pH dependence of DGL shows an optimum at 7.5-8.5, suggesting a requirement for a catalytic base. α-Glycerophosphate and inorganic phosphate are weak competitive inhibitors, with Ki values near 30mM, and d-serine is neither a substrate nor an inhibitor. We have found in rapid-scanning stopped-flow experiments that DGL reacts rapidly with its substrate to form a quinonoid intermediate with λmax=480nm, within the dead time (ca. 2msec), which then rapidly decays (k=279s-1) to an intermediate with absorption between 330 and 350nm, probably an aminoacrylate complex. We suggest a mechanism for DGL and propose that the unusual stereochemistry of the DGL reaction requires a catalytic base poised on the opposite face of the PLP-substrate complex from the other members of the aminotransferase superfamily.