Abstract
l‐Amino acid oxidase/monooxygenase from Pseudomonas sp. AIU 813 (l‐AAO/MOG) catalyzes both the oxidative deamination and oxidative decarboxylation of the α‐group of l‐Lys to produce a keto acid and amide, respectively. l‐AAO/MOG exhibits limited specificity for l‐amino acid substrates with a basic side chain. We previously determined its ligand‐free crystal structure and identified a key residue for maintaining the dual activities. Here, we determined the structures of l‐AAO/MOG complexed with l‐Lys, l‐ornithine, and l‐Arg and revealed its substrate recognition. Asp238 is located at the ceiling of a long hydrophobic pocket and forms a strong interaction with the terminal, positively charged group of the substrates. A mutational analysis on the D238A mutant indicated that the interaction is critical for substrate binding but not for catalytic control between the oxidase/monooxygenase activities. The catalytic activities of the D238E mutant unexpectedly increased, while the D238F mutant exhibited altered substrate specificity to long hydrophobic substrates. In the ligand‐free structure, there are two channels connecting the active site and solvent, and a short region located at the dimer interface is disordered. In the l‐Lys complex structure, a loop region is displaced to plug the channels. Moreover, the disordered region in the ligand‐free structure forms a short helix in the substrate complex structures and creates the second binding site for the substrate. It is assumed that the amino acid substrate enters the active site of l‐AAO/MOG through this route.DatabaseThe atomic coordinates and structure factors (codes 5YB6, 5YB7, and 5YB8) have been deposited in the Protein Data Bank (http://wwpdb.org/).EC numbers 1.4.3.2 (l‐amino acid oxidase), 1.13.12.2 (lysine 2‐monooxygenase).
Highlights
The structures of L-AAO/MOG complexed with either L-Lys, L-Orn, or L-Arg were determined at 2.0–2.3 A resolution (Table 1)
When yellow L-AAO/MOG crystals were soaked in the crystallization buffer containing the substrate (L-Lys, L-Orn, or L-Arg), the color changed to pale yellow, suggesting that the flavin cofactor was partially reduced
In contrast to common amino acid oxidases that act on the a-amino group, LLys e-oxidase (LodA) is a cysteine tryptophylquinone-dependent enzyme and employs a completely different system for the recognition and catalysis of the oxidase reaction of the side chain e-amino group
Summary
AIU 813 (L-AAO/MOG) catalyzes both the oxidative deamination and oxidative decarboxylation of the a-group of L-Lys to produce a keto acid and amide, respectively. L-AAO/MOG exhibits limited specificity for L-amino acid substrates with a basic side chain. We determined the structures of L-AAO/MOG complexed with L-Lys, L-ornithine, and L-Arg and revealed its substrate recognition. In the ligand-free structure, there are two channels connecting the active site and solvent, and a short region located at the dimer interface is disordered. The disordered region in the ligand-free structure forms a short helix in the substrate complex structures and creates the second binding site for the substrate. It is assumed that the amino acid substrate enters the active site of L-AAO/MOG through this route.
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