L-amino-acid oxidase

 

L-aminoacid oxidase is a dimeric flavoprotein. it uses a non-covalently bound FAD cofactor in catalysing the stereospecific oxidative deamination of an L-amino acid substrate to an alpha ketoacid, forming ammonia and hydrogen peroxide. The enzyme shows a marked preference for hydrophobic amino acids including phenylalanine, tryptophan, tyrosine and leucine.

 

Reference Protein and Structure

Sequence
P81382 UniProt (1.4.3.2) IPR002937 (Sequence Homologues) (PDB Homologues)
Biological species
Calloselasma rhodostoma (Malayan pit viper) Uniprot
PDB
1f8r - CRYSTAL STRUCTURE OF L-AMINO ACID OXIDASE FROM CALLOSELASMA RHODOSTOMA COMPLEXED WITH CITRATE (2.0 Å) PDBe PDBsum 1f8r
Catalytic CATH Domains
3.90.660.10 CATHdb 1.10.405.10 CATHdb (see all for 1f8r)
Cofactors
Fadh2(2-) (1)
Click To Show Structure

Enzyme Reaction (EC:1.4.3.2)

water
CHEBI:15377ChEBI
+
dioxygen
CHEBI:15379ChEBI
+
L-alpha-amino acid zwitterion
CHEBI:59869ChEBI
→
ammonium
CHEBI:28938ChEBI
+
hydrogen peroxide
CHEBI:16240ChEBI
+
2-oxo monocarboxylic acid anion
CHEBI:35179ChEBI
Alternative enzyme names: Ophio-amino-acid oxidase,

Enzyme Mechanism

Introduction

The non-covalently bound FAD is first reduced to FADH2 with oxidation of the amino acid to an imino acid. Non-enzymatic hydrolysis at the imine centre leads to the elimination of ammonia and formation of an alpha-ketoacid. The mechanism is thought to involve a base for the abstraction of a proton from the amino group of the substrate and subsequent transfer of a hydride from the alpha carbon to the N5 of the isoalloxazine ring of FAD to form the imino intermediate.

Catalytic Residues Roles

UniProt PDB* (1f8r)
His241 His223A The residue is thought to act as a general base towards the zwitter-ionic form of the substrate, abstracting a proton from the amino group and so activating the substrate to transfer a hydride to the FAD cofactor. proton acceptor, proton donor
Lys344 Lys326A The residue acts to relay a proton to a structurally conserved water molecule, which is thought to assist peroxide formation from the flavin-hydroperoxy intermediate. proton acceptor
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

proton transfer, overall reactant used, aromatic bimolecular nucleophilic addition, hydride transfer, intermediate formation, bimolecular nucleophilic addition, elimination (not covered by the Ingold mechanisms), overall product formed, native state of cofactor regenerated

References

  1. Moustafa IM et al. (2006), J Mol Biol, 364, 991-1002. Crystal Structure of LAAO from Calloselasma rhodostoma with an l-Phenylalanine Substrate: Insights into Structure and Mechanism. DOI:10.1016/j.jmb.2006.09.032. PMID:17046020.
  2. Pawelek PD et al. (2000), EMBO J, 19, 4204-4215. The structure of L-amino acid oxidase reveals the substrate trajectory into an enantiomerically conserved active site. DOI:10.1093/emboj/19.16.4204. PMID:10944103.

Step 1. His223 acts as a base to deprotonate the amine group of the amino acid substrate.

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Catalytic Residues Roles

Residue Roles
His223A proton acceptor

Chemical Components

proton transfer, overall reactant used

Step 2. Once depronated the substrate is activated for hydride transfer from the alpha-C atom to the N5 of FAD, forming the imine intermediate.

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Catalytic Residues Roles

Residue Roles

Chemical Components

ingold: aromatic bimolecular nucleophilic addition, hydride transfer, intermediate formation

Step 3. The presence of a structurally conserved water molecule and lysine residue suggest a possible role for Lys326 in activating the water for hydrolysis of the intermediate. The intermediate is subject to nucleophilic attack by the water, while the imine accepts a proton.

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Catalytic Residues Roles

Residue Roles
His223A proton donor
Lys326A proton acceptor

Chemical Components

ingold: bimolecular nucleophilic addition, proton transfer

Step 4. In an inferred hydrolysis step there is movement of electrons from the hydroxyl lone pair, causing elimination of ammonia and forming the product. Following hydrolysis there is an oxidative half reaction in which the cofactor FADH- is reoxidised to FAD by molecular oxygen.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles

Chemical Components

elimination (not covered by the Ingold mechanisms), overall product formed, native state of cofactor regenerated
Download: Image, Marvin File

Step 1. His223 acts as a base to deprotonate the amine group of the amino acid substrate.

Step 2. Once depronated the substrate is activated for hydride transfer from the alpha-C atom to the N5 of FAD, forming the imine intermediate.

Step 3. The presence of a structurally conserved water molecule and lysine residue suggest a possible role for Lys326 in activating the water for hydrolysis of the intermediate. The intermediate is subject to nucleophilic attack by the water, while the imine accepts a proton.

Step 4. In an inferred hydrolysis step there is movement of electrons from the hydroxyl lone pair, causing elimination of ammonia and forming the product. Following hydrolysis there is an oxidative half reaction in which the cofactor FADH- is reoxidised to FAD by molecular oxygen.

Products.

Contributors

James W. Murray, Craig Porter, Gemma L. Holliday, Amelia Brasnett