The Structure And Mechanism Of Amino Acid Dehydrogenase AADH

The 3,5-diaminohexanoate dehydrogenase(AADH)from Candidatus Cloacamonas acidaminovorans is an NAD(P)H-dependent enzyme which catalyzes the reductive amination of 3-keto-5-aminohexanoate to(3S,5S)-diaminohexanoate((3S,5S)-DAH).The wildtype enzyme has strict substrate specificity toward(3S,5S)-DAH and has no activity toward other ?-amino substrates.However,the double mutant E310G/A314 Y could expand the substrate spectrum to(R)-?-homo Met,(R)-?-Phe,(S)-?-homo Tyr and(S)-?-AB.To probe the molecular mechanism of substrate recognition,we purified and crystalized both the wildtype AADH the mutant E310G/A314,and determined their crystal structures(2.3 ? resolution for wildtype AADH and 2.48 ? resolution respectively.)By comparing these two structures,we found Glu310 forms intermolecular hydrogen bond with the residues in the adjacent chain and therefore prevent the corresponding loop from moving.The conformation of this loop in mutant E310G/A314 Y is much more flexible than that in wildtype AADH.We further determined the complex structure of the mutant E310G/A314 and NADP at 3.0 ?)resolution.We found that Glu310 in one chain positioned in the active site of the adjacent chain.By docking the substrate in the above complex structure,we found that the hydrogen bonding network among the NADP,the substrate and the Glu 310 on the adjacent chain,provide the position of the substrate.The size of the substrate 3,5-diaminohexanoic acid just matches the active pocket.Residues Asp117 and Met96 keep the bulky ?-Phe far away from NADP and thus prevent it from participating the reaction.In the mutant E310G/A314 Y,the hydrogen bond network is greatly weakened,and the catalytic loop becomes more flexible.This explain why the mutant E310G/A314 Y has broad substrate selectivity.