D-amino-acid oxidase

 

D amino acid oxidase (DAAO) acts to oxidise D amino acids to their imino counterparts, using FAD as a cofactor. The imino acid then undergoes hydrolysis outside of the enzyme's active site. This reaction has been found to play an important role in the degradation of certain neurotransmitters such as D-serine in mammals, but the enzyme is also found in other eukaryotes and its function has not been fully characterised. It shows mechanistic correlation with other enzymes able to oxidise amino groups.

Yeast and mammalian DAAOs share features such as the basic catalytic mechanism. However, they differ in important aspects such as catalytic efficiency, substrate specificity, aggregation state, stability, kinetic mechanism, and mode and effectiveness of FAD binding. Thus, DAAO from the yeast Rhodotorula gracilis (RgDAAO) has a kcat value ≈20,000 minute−1 compared to 600 minute−1 for pig kidney DAAO (pkDAAO) with D-alanine as substrate.

 

Reference Protein and Structure

Sequence
P80324 UniProt (1.4.3.3) IPR023209 (Sequence Homologues) (PDB Homologues)
Biological species
Rhodotorula toruloides (Yeast) Uniprot
PDB
1c0p - D-AMINO ACIC OXIDASE IN COMPLEX WITH D-ALANINE AND A PARTIALLY OCCUPIED BIATOMIC SPECIES (1.2 Å) PDBe PDBsum 1c0p
Catalytic CATH Domains
3.40.50.720 CATHdb (see all for 1c0p)
Cofactors
Fadh2(2-) (1)
Click To Show Structure

Enzyme Reaction (EC:1.4.3.3)

D-alpha-amino acid zwitterion
CHEBI:59871ChEBI
+
dioxygen
CHEBI:15379ChEBI
+
water
CHEBI:15377ChEBI
→
ammonium
CHEBI:28938ChEBI
+
2-oxo monocarboxylic acid anion
CHEBI:35179ChEBI
+
hydrogen peroxide
CHEBI:16240ChEBI
Alternative enzyme names: L-amino acid:O(2) oxidoreductase, New yellow enzyme, Ophio-amino-acid oxidase,

Enzyme Mechanism

Introduction

The mechanism of the reaction is through direct hydride transfer from the alpha carbon of the amino acid to the N5 of the FAD, with concomitant deprotonation of the alpha amino group by Ser 335's side chain allowing orbital overlap between the nitrogen lone pair and the carbon's sigma* orbital to occur, resulting in the imine product. The transition state for this process is planar and stabilised by hydrogen bonding between the carbonyl of Ser 335 and the amino group. This mechanism is supported by high resolution crystal structures, kinetic isotope effects and free energy correlation calculations [PMID:11070076].

Catalytic Residues Roles

UniProt PDB* (1c0p)
Ser335 (main-C), Gln339 (main-C), Asn54 (main-N), Asn54 (main-C) Ser1335(337)A (main-C), Gln1339(341)A (main-C), Asn1054(56)A (main-N), Asn1054(56)A (main-C) Acts to lower the pKa of the amino acid substrate. modifies pKa, hydrogen bond acceptor, hydrogen bond donor
Ser335 Ser1335(337)A Acts as a general acid/base. proton relay, proton acceptor, proton donor
*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

bimolecular elimination, hydride transfer, aromatic bimolecular nucleophilic addition, overall reactant used, cofactor used, intermediate formation, overall product formed, proton relay, rate-determining step, radical formation, electron transfer, radical termination, bimolecular homolytic addition, proton transfer, aromatic intramolecular elimination, native state of cofactor regenerated, intermediate terminated, native state of enzyme regenerated, reaction occurs outside the enzyme, bimolecular nucleophilic addition, deamination, intramolecular elimination

References

  1. Umhau S et al. (2000), Proc Natl Acad Sci U S A, 97, 12463-12468. The x-ray structure of D-amino acid oxidase at very high resolution identifies the chemical mechanism of flavin-dependent substrate dehydrogenation. DOI:10.1073/pnas.97.23.12463. PMID:11070076.
  2. Ghisla S et al. (2011), J Biol Chem, 286, 40987-40998. Revisitation of the  Cl-Elimination Reaction of D-Amino Acid Oxidase: NEW INTERPRETATION OF THE REACTION THAT SPARKED FLAVOPROTEIN DEHYDROGENATION MECHANISMS. DOI:10.1074/jbc.m111.266536. PMID:21949129.
  3. Rosini E et al. (2011), FEBS J, 278, 482-492. On the reaction of d-amino acid oxidase with dioxygen: O2 diffusion pathways and enhancement of reactivity. DOI:10.1111/j.1742-4658.2010.07969.x. PMID:21182588.
  4. Katane M et al. (2008), Amino Acids, 35, 75-82. Hyperactive mutants of mouse d-aspartate oxidase: mutagenesis of the active site residue serine 308. DOI:10.1007/s00726-007-0627-8. PMID:18235994.
  5. Boselli A et al. (2007), Biochimie, 89, 360-368. Investigating the role of active site residues of Rhodotorula gracilis d-amino acid oxidase on its substrate specificity. DOI:10.1016/j.biochi.2006.10.017. PMID:17145127.
  6. Caldinelli L et al. (2006), FEBS J, 273, 504-512. Tryptophan 243 affects interprotein contacts, cofactor binding and stability in D-amino acid oxidase from Rhodotorula gracilis. DOI:10.1111/j.1742-4658.2005.05083.x. PMID:16420474.
  7. Tishkov VI et al. (2005), Biochemistry (Mosc), 70, 40-54. D-amino acid oxidase: structure, catalytic mechanism, and practical application. DOI:10.1007/s10541-005-0050-2.
  8. Boselli A et al. (2004), Biochim Biophys Acta, 1702, 19-32. On the mechanism of Rhodotorula gracilis d-amino acid oxidase: role of the active site serine 335. DOI:10.1016/j.bbapap.2004.07.005. PMID:15450847.
  9. Pollegioni L et al. (2004), Biotechnol Prog, 20, 467-473. Catalytic Properties of d-Amino Acid Oxidase in Cephalosporin C Bioconversion: A Comparison between Proteins from Different Sources. DOI:10.1021/bp034206q. PMID:15058991.
  10. Pollegioni L et al. (2002), J Mol Biol, 324, 535-546. Yeast d-Amino Acid Oxidase: Structural Basis of its Catalytic Properties. DOI:10.1016/s0022-2836(02)01062-8.
  11. Pilone MS (2000), Cell Mol Life Sci, 57, 1732-1747. D-Amino acid oxidase: new findings. DOI:10.1007/pl00000655. PMID:11130179.

Step 1. Water deprotonates Ser1335, which deprotonates the amine of the D-amino acid, eliminating a hydride ion, which adds to FAD.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ser1335(337)A (main-C) hydrogen bond acceptor, hydrogen bond donor
Asn1054(56)A (main-N) hydrogen bond donor, hydrogen bond acceptor
Gln1339(341)A (main-C) hydrogen bond acceptor
Asn1054(56)A (main-N) modifies pKa
Asn1054(56)A (main-C) modifies pKa
Ser1335(337)A (main-C) modifies pKa
Gln1339(341)A (main-C) modifies pKa
Ser1335(337)A proton donor, proton relay, proton acceptor

Chemical Components

ingold: bimolecular elimination, hydride transfer, ingold: aromatic bimolecular nucleophilic addition, overall reactant used, cofactor used, intermediate formation, overall product formed, proton relay, rate-determining step

Step 2. The FAD undergoes double bond rearrangement which causes a single electron to be transferred to a dioxygen molecule.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles

Chemical Components

radical formation, electron transfer, overall reactant used, intermediate formation

Step 3. The dioxygen molecule undergoes a homolytic reaction in which it colligates to FAD, with concomitant deprotonation of water.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn1054(56)A (main-N) hydrogen bond donor, hydrogen bond acceptor
Gln1339(341)A (main-C) hydrogen bond acceptor

Chemical Components

radical termination, ingold: bimolecular homolytic addition, proton transfer, intermediate formation

Step 4. The peroxo group deprotonates FAD, which initiates the elimination of hydrogen peroxide.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn1054(56)A (main-N) hydrogen bond donor, hydrogen bond acceptor
Gln1339(341)A (main-C) hydrogen bond acceptor

Chemical Components

ingold: aromatic intramolecular elimination, native state of cofactor regenerated, intermediate terminated, overall product formed, native state of enzyme regenerated

Step 5. The product of the enzyme undergoes spontaneous hydrolysis outside of the active site to produce ammonium and the 2-oxo acid.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles

Chemical Components

reaction occurs outside the enzyme, proton transfer, ingold: bimolecular nucleophilic addition

Step 6. Second half of the spontaneous hydrolysis of ammonia and the 2-oxo acid.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles

Chemical Components

deamination, reaction occurs outside the enzyme, ingold: intramolecular elimination, proton transfer
Download: Image, Marvin File

Step 1. Water deprotonates Ser1335, which deprotonates the amine of the D-amino acid, eliminating a hydride ion, which adds to FAD.

Step 2. The FAD undergoes double bond rearrangement which causes a single electron to be transferred to a dioxygen molecule.

Step 3. The dioxygen molecule undergoes a homolytic reaction in which it colligates to FAD, with concomitant deprotonation of water.

Step 4. The peroxo group deprotonates FAD, which initiates the elimination of hydrogen peroxide.

Step 5. The product of the enzyme undergoes spontaneous hydrolysis outside of the active site to produce ammonium and the 2-oxo acid.

Step 6. Second half of the spontaneous hydrolysis of ammonia and the 2-oxo acid.

Products.

Introduction

A carbanion mechanism has been proposed in which an enzyme base removes the alpha-proton (or possibly the amino proton) and so forms a carbanion intermediate. The resulting unstable carbanion would rapidly attack the N-5 locus of the flavin. Subsequent rapid rearrangement would result in reduced flavin and iminopyruvate. This mechanism was suggested upon the observation that pig kidney DAAO catalyses the elimination of halide from beta-halogenated amino acids.

Catalytic Residues Roles

UniProt PDB* (1c0p)
Ser335 (main-C), Gln339 (main-C), Asn54 (main-N), Asn54 (main-C) Ser1335(337)A (main-C), Gln1339(341)A (main-C), Asn1054(56)A (main-N), Asn1054(56)A (main-C) Act to lower the pKa of the alpha carbon proton. modifies pKa
Ser335 Ser1335(337)A Acts as a general acid/base. proton relay, proton acceptor, proton donor
*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, hydride transfer, cofactor used, radical formation, electron transfer, overall reactant used, intermediate formation, radical termination, bimolecular homolytic addition, aromatic intramolecular elimination, native state of cofactor regenerated, intermediate terminated, overall product formed, native state of enzyme regenerated, bimolecular nucleophilic addition, reaction occurs outside the enzyme, intramolecular elimination, deamination

References

  1. Walsh CT et al. (1971), J Biol Chem, 246, 6855-6866. Studies on the mechanism of action of D-amino acid oxidase. Evidence for removal of substrate -hydrogen as a proton. PMID:4399475.
  2. Ghisla S et al. (2011), J Biol Chem, 286, 40987-40998. Revisitation of the  Cl-Elimination Reaction of D-Amino Acid Oxidase: NEW INTERPRETATION OF THE REACTION THAT SPARKED FLAVOPROTEIN DEHYDROGENATION MECHANISMS. DOI:10.1074/jbc.m111.266536. PMID:21949129.
  3. Harris CM et al. (2001), Eur J Biochem, 268, 5504-5520. pH and kinetic isotope effects in d-amino acid oxidase catalysis. PMID:11683874.

Step 1. Ser1335 abstracts a proton from the alpha carbon, forming a carbanion intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn1054(56)A (main-N) modifies pKa
Asn1054(56)A (main-C) modifies pKa
Ser1335(337)A (main-C) modifies pKa
Gln1339(341)A (main-C) modifies pKa
Ser1335(337)A proton donor, proton relay, proton acceptor

Chemical Components

proton transfer

Step 2. The resulting unstable carbanion undergoes rearrangement to produce the reduced flavin and iminopyruvate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn1054(56)A (main-N) modifies pKa
Asn1054(56)A (main-C) modifies pKa
Ser1335(337)A (main-C) modifies pKa
Gln1339(341)A (main-C) modifies pKa

Chemical Components

hydride transfer, cofactor used

Step 3. The FAD undergoes double bond rearrangement which causes a single electron to be transferred to a dioxygen molecule.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles

Chemical Components

radical formation, electron transfer, overall reactant used, intermediate formation

Step 4. The dioxygen molecule undergoes a homolytic reaction in which it colligates to FAD, with concomitant deprotonation of water.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn1054(56)A (main-N) hydrogen bond donor, hydrogen bond acceptor
Gln1339(341)A (main-C) hydrogen bond acceptor

Chemical Components

radical termination, ingold: bimolecular homolytic addition, proton transfer, intermediate formation

Step 5. The peroxo group deprotonates FAD, which initiates the elimination of hydrogen peroxide.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn1054(56)A (main-N) hydrogen bond donor, hydrogen bond acceptor
Gln1339(341)A (main-C) hydrogen bond acceptor

Chemical Components

ingold: aromatic intramolecular elimination, native state of cofactor regenerated, intermediate terminated, overall product formed, native state of enzyme regenerated

Step 6. The product of the enzyme undergoes spontaneous hydrolysis outside of the active site to produce ammonium and the 2-oxo acid.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles

Chemical Components

ingold: bimolecular nucleophilic addition, proton transfer, reaction occurs outside the enzyme

Step 7. Second half of the spontaneous hydrolysis of ammonia and the 2-oxo acid.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles

Chemical Components

proton transfer, ingold: intramolecular elimination, reaction occurs outside the enzyme, deamination
Download: Image, Marvin File

Step 1. Ser1335 abstracts a proton from the alpha carbon, forming a carbanion intermediate.

Step 2. The resulting unstable carbanion undergoes rearrangement to produce the reduced flavin and iminopyruvate.

Step 3. The FAD undergoes double bond rearrangement which causes a single electron to be transferred to a dioxygen molecule.

Step 4. The dioxygen molecule undergoes a homolytic reaction in which it colligates to FAD, with concomitant deprotonation of water.

Step 5. The peroxo group deprotonates FAD, which initiates the elimination of hydrogen peroxide.

Step 6. The product of the enzyme undergoes spontaneous hydrolysis outside of the active site to produce ammonium and the 2-oxo acid.

Step 7. Second half of the spontaneous hydrolysis of ammonia and the 2-oxo acid.

Products.

Contributors

Gemma L. Holliday, Daniel E. Almonacid, Peter Sarkies