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Mechanistic Studies of an Amine Oxidase Derived from d-Amino Acid Oxidase.

ABSTRACT: The flavoprotein d-amino acid oxidase has long served as a paradigm for understanding the mechanism of oxidation of amino acids by flavoproteins. Recently, a mutant d-amino acid oxidase (Y228L/R283G) that catalyzed the oxidation of amines rather than amino acids was described [Yasukawa, K., et al. (2014) Angew. Chem., Int. Ed. 53, 4428-4431]. We describe here the use of pH and kinetic isotope effects with (R)-?-methylbenzylamine as a substrate to determine whether the mutant enzyme utilizes the same catalytic mechanism as the wild-type enzyme. The effects of pH on the steady-state and rapid-reaction kinetics establish that the neutral amine is the substrate, while an active-site residue, likely Tyr224, must be uncharged for productive binding. There is no solvent isotope effect on the kcat/Km value for the amine, consistent with the neutral amine being the substrate. The deuterium isotope effect on the kcat/Km value is pH-independent, with an average value of 5.3, similar to values found with amino acids as substrates for the wild-type enzyme and establishing that there is no commitment to catalysis with this substrate. The kcat/KO2 value is similar to that seen with amino acids as the substrate, consistent with the oxidative half-reaction being unperturbed by the mutation and with flavin oxidation preceding product release. All of the data are consistent with the mutant enzyme utilizing the same mechanism as the wild-type enzyme, transfer of hydride from the neutral amine to the flavin.

SUBMITTER: Trimmer EE

PROVIDER: S-EPMC5472355 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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Mechanistic Studies of an Amine Oxidase Derived from d-Amino Acid Oxidase.

Trimmer Elizabeth E EE Wanninayake Udayanga S US Fitzpatrick Paul F PF

Biochemistry 20170403 14

The flavoprotein d-amino acid oxidase has long served as a paradigm for understanding the mechanism of oxidation of amino acids by flavoproteins. Recently, a mutant d-amino acid oxidase (Y228L/R283G) that catalyzed the oxidation of amines rather than amino acids was described [Yasukawa, K., et al. (2014) Angew. Chem., Int. Ed. 53, 4428-4431]. We describe here the use of pH and kinetic isotope effects with (R)-α-methylbenzylamine as a substrate to determine whether the mutant enzyme utilizes the ...[more]

PMID: 28355481

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Project description:BackgroundSchizophrenia is a neurodegenerative disorder that occurs worldwide and can be difficult to diagnose. It is the foremost neurological disorder leading to suicide among patients in both developed and underdeveloped countries. D-amino acid oxidase activator (DAOA), also known as G72, is directly implicated in the glutamateric hypothesis of schizophrenia. It activates D-amino acid oxidase, which oxidizes D-serine, leading to modulation of the N-methyl-D-aspartate receptor.MethodsMODELLER (9v10) was utilized to generate three dimensional structures of the DAOA candidate gene. The HOPE server was used for mutational analysis. The Molecular Evolutionary Genetics Analysis (MEGA5) tool was utilized to reconstruct the evolutionary history of the candidate gene DAOA. AutoDock was used for protein-ligand docking and Gramm-X and PatchDock for protein-protein docking.ResultsA suitable template (1ZCA) was selected by employing BLASTp on the basis of 33% query coverage, 27% identity and E-value 4.9. The Rampage evaluation tool showed 91.1% favored region, 4.9% allowed region and 4.1% outlier region in DAOA. ERRAT demonstrated that the predicted model had a 50.909% quality factor. Mutational analysis of DAOA revealed significant effects on hydrogen bonding and correct folding of the DAOA protein, which in turn affect protein conformation. Ciona was inferred as the outgroup. Tetrapods were in their appropriate clusters with bifurcations. Human amino acid sequences are conserved, with chimpanzee and gorilla showing more than 80% homology and bootstrap value based on 1000 replications. Molecular docking analysis was employed to elucidate the binding mode of the reported ligand complex for DAOA. The docking experiment demonstrated that DAOA is involved in major amino acid interactions: the residues that interact most strongly with the ligand C28H28N3O5PS2 are polar but uncharged (Gln36, Asn38, Thr 122) and non-polar hydrophobic (Ile119, Ser171, Ser21, Ala31). Protein-protein docking simulation demonstrated two ionic bonds and one hydrogen bond involving DAOA. Lys-7 of the receptor protein interacted with Lys-163 and Asp-2037. Tyr-03 interacted with Arg-286 of the ligand protein and formed a hydrogen bond.ConclusionThe predicted interactions might serve to inhibit the disease-related allele. It is assumed that current bioinformatics methods will contribute significantly to identifying, analyzing and curing schizophrenia. There is an urgent need to develop effective drugs for schizophrenia, and tools for examining candidate genes more accurately and efficiently are required.

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Mechanistic Studies of an Amine Oxidase Derived from d-Amino Acid Oxidase.

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Mechanistic Studies of an Amine Oxidase Derived from d-Amino Acid Oxidase.

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