Ligand complex structures of l‐amino acid oxidase/monooxygenase from Pseudomonas sp. AIU 813 and its conformational change

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. Database The 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).

Catalytic mechanism of ancestral L-lysine oxidase assigned by sequence data mining

“…From a structural comparison between the ligand-free form and the L-Lys or L-Arg binding form of AncLLysO, substrate binding appeared to induce a conformational change of a loop which is formed by residues 251-254 (251-GGYY-254); cis-trans transformation at G251 is remarkable in this change. The loop position is consistent with a plug loop in L-LOX/MOG, which occurs when these two structures are superimposed on each other (40). Therefore, we call the loop a "plug loop" as well.…”

“…Referring to the previous study (40), enzyme kinetics for the monooxygenase activity of AncLLysO were assayed utilizing the purified AcAOx as the quantifying enzymes of 5-APNM. However, the concentration of 5-APNM could not be quantified by the assay because the amount of 5-APNM produced by AncLLysO was too low to detect with AcAOx.…”

“…The assigned LLysO has low sequence identity (less than 30%) to the previously reported LLysO from Trichoderma viridae (TvLysOX) (37,38) and L-amino acid oxidase/monooxygenase (L-LOX/MOG) from Pseudomonas sp. AIU813 (39)(40)(41); therefore, the enzymatic properties of the assigned LLysO could not be estimated only from the sequence analysis. Ancestral LLysO (AncLLysO) was designed utilizing the assigned LLysO sequences as templates.…”

Structural basis of strict substrate recognition of l‐lysine α‐oxidase from Trichoderma viride

“…AIU 813 has been determined. 20 L-AAO/MOG shows strict substrate specificity for L-lysine, similar to LysOX but rather high relative activities for other positively charged L-amino acids, such as L-arginine and L-ornithine, compared with LysOX. L-AAO/MOG has an aspartic acid (D238) that directly interacts with ε-amino group of L-lysine in the substrate-binding pocket.…”

“…Recently, the ligand complex structure of LAAO /monooxygenase (MOG) from Pseudomonas sp. AIU 813 has been determined 20 . L‐AAO/MOG shows strict substrate specificity for l ‐lysine, similar to LysOX but rather high relative activities for other positively charged l ‐amino acids, such as l ‐arginine and l ‐ornithine, compared with LysOX.…”