Structure of FAD-Bound l-Aspartate Oxidase: Insight into Substrate Specificity and Catalysis,

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Abstract
L-Aspartate oxidase (Laspo) catalyzes the conversion of l-Asp to iminoaspartate, the first step in the de novo biosynthesis of NAD+. This bacterial pathway represents a potential drug target since it is absent in mammals. The Laspo R386L mutant was crystallized in the FAD-bound catalytically competent form and its three-dimensional structure determined at 2.5 Å resolution in both the native state and in complex with succinate. Comparison of the R386L holoprotein with the wild-type apoenzyme [Mattevi, A., Tedeschi, G., Bacchella, L., Coda, A., Negri, A., and Ronchi, S. (1999) Structure7, 745−756] reveals that cofactor incorporation leads to the ordering of two polypeptide segments (residues 44−53 and 104−141) and to a 27° rotation of the capping domain. This motion results in the formation of the active site cavity, located at the interface between the capping domain and the FAD-binding domain. The structure of the succinate complex indicates that the cavity surface is decorated by two clusters of H-bond donors that anchor the ligand carboxylates. Moreover, Glu121, which is strictly conserved among Laspo sequences, is positioned to interact with the l-Asp α-amino group. The architecture of the active site of the Laspo holoenzyme is remarkably similar to that of respiratory fumarate reductases, providing strong evidence for a common mechanism of catalysis in Laspo and flavoproteins of the succinate dehydrogenase/fumarate reductase family. This implies that Laspo is mechanistically distinct from other flavin-dependent amino acid oxidases, such as the prototypical d-amino acid oxidase.