D-arginine dehydrogenase

 

D-arginine dehydrogenase (DADH) is a flavin-dependent oxidoreductase. It is a member of the structural family consisting of D-amino acid oxidase, sarcosine oxidase, dimethyl glycine oxidase, and glycine oxidase. The enzyme has broad substrate specificity towards D-amino acids, particularly with cationic and hydrophobic D-amino acids. DADH and L-arginine dehydrogenase (LADH) make up a two-enzyme system involved in D- to L-arginine racemisation in pseudomonads and related species and both enzymes are products of the dauBAR operon, by D-arginine and D-lysine.

Crystallographic data shows that DADH from Pseudomonas aeruginosa (PaDADH) contains an active site loop L1 with two flexible segments that span the FAD-binding site and the substrate-binding site. It can occupy two distinct conformations, an open conformation competent for substrate binding and a closed conformation which is catalytically relevant and ligand-bound.

 

Reference Protein and Structure

Sequence
Q9HXE3 UniProt (1.4.99.6) IPR036188 (Sequence Homologues) (PDB Homologues)
Biological species
Pseudomonas aeruginosa PAO1 (Bacteria) Uniprot
PDB
3nye - Crystal Structure of Pseudomonas aeruginosa D-Arginine Dehydrogenase in Complex with Imino-Arginine (1.3 Å) PDBe PDBsum 3nye
Catalytic CATH Domains
3.30.9.10 CATHdb 3.50.50.60 CATHdb (see all for 3nye)
Click To Show Structure

Enzyme Reaction (EC:1.4.99.6)

FAD2-
CHEBI:X00681X00681
+
water
CHEBI:15377ChEBI
+
D-argininium(1+)
CHEBI:32689ChEBI
→
hydron
CHEBI:15378ChEBI
+
ammonium
CHEBI:28938ChEBI
+
5-guanidino-2-oxopentanoic acid zwitterion
CHEBI:58489ChEBI
+
FADH3-
CHEBI:X00682X00682
Alternative enzyme names: D-amino-acid:(acceptor) oxidoreductase (deaminating), D-amino-acid dehydrogenase, D-amino-acid:acceptor oxidoreductase (deaminating),

Enzyme Mechanism

Introduction

Hydride transfer mechanism supported by kinetic studies. In principle, transfer of an electron and a hydrogen atom from the substrate to the flavin is possible, but there is no evidence for presence of a radical as of yet. Deprotonation of the substrate α-amine occurs first, triggering CH bond cleavage through hydride transfer to the flavin N5 atom. Subsequent hydrolysis forms 5-guanidino-2-oxopentanoic acid and ammonia.

Catalytic Residues Roles

UniProt PDB* (3nye)
Tyr53, Tyr249 Tyr1053(59)A, Tyr1249(255)A Stabilise transition state in PaDADH to facilitate the hydride transfer mechanism. transition state stabiliser
Glu87 Glu1087(93)A Stabilises D-arginine by engaging in an ionic interaction with the substrate guanido group. electrostatic stabiliser
His48 His1048(54)A His48 forms a hydrogen-bond network with the amino group of the substrate, separated by two water molecules. The network is important to optimise substrate binding and prevent a slow proton release from the enzyme-substrate complex. increase acidity
Ser45, Ala46 Ser1045(51)A, Ala1046(52)A In the FAD-binding site the side chains of S45 and A46 in loop L1, which do not interact directly with the substrate, adopt two alternate conformations depending on whether the ligand is present in the active site or not. steric role
*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, intermediate formation, reaction occurs outside the enzyme, hydrolysis, bimolecular nucleophilic addition, intramolecular elimination, overall product formed

References

  1. Gannavaram S et al. (2014), Biochemistry, 53, 6574-6583. Mechanistic and computational studies of the reductive half-reaction of tyrosine to phenylalanine active site variants of D-arginine dehydrogenase. DOI:10.1021/bi500917q. PMID:25243743.
  2. Ouedraogo D et al. (2017), Arch Biochem Biophys, 632, 192-201. Amine oxidation by d-arginine dehydrogenase in Pseudomonas aeruginosa. DOI:10.1016/j.abb.2017.06.013. PMID:28625766.
  3. Ball J et al. (2015), Arch Biochem Biophys, 568, 56-63. Importance of glutamate 87 and the substrate α-amine for the reaction catalyzed by D-arginine dehydrogenase. DOI:10.1016/j.abb.2015.01.017. PMID:25637657.
  4. Yuan H et al. (2011), J Am Chem Soc, 133, 18957-18965. Insights on the mechanism of amine oxidation catalyzed by D-arginine dehydrogenase through pH and kinetic isotope effects. DOI:10.1021/ja2082729. PMID:21999550.
  5. Fu G et al. (2011), Biochemistry, 50, 6292-6294. Atomic-resolution structure of an N5 flavin adduct in D-arginine dehydrogenase. DOI:10.1021/bi200831a. PMID:21707047.

Step 1. Deprotonation of the substrate α-amine, triggering CH bond cleavage in the next step.

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Catalytic Residues Roles

Residue Roles
Glu1087(93)A electrostatic stabiliser
His1048(54)A increase acidity
Ser1045(51)A steric role
Ala1046(52)A steric role

Chemical Components

proton transfer

Step 2. CH bond cleavage through the transfer of a hydride ion from the substrate to the flavin N5 atom.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Tyr1053(59)A transition state stabiliser
Tyr1249(255)A transition state stabiliser
Glu1087(93)A electrostatic stabiliser

Chemical Components

hydride transfer, cofactor used, intermediate formation

Step 3. Non-enzymatic hydrolysis of intermediate iminoacid to α-keto acid and ammonia. Begins with the addition of water.

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Catalytic Residues Roles

Residue Roles

Chemical Components

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

Step 4. Proton transfer.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles

Chemical Components

reaction occurs outside the enzyme, hydrolysis, proton transfer

Step 5. Elimination of ammonia.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles

Chemical Components

reaction occurs outside the enzyme, hydrolysis, ingold: intramolecular elimination

Step 6. Deprotonation to form final product.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles

Chemical Components

reaction occurs outside the enzyme, hydrolysis, proton transfer, overall product formed
Download: Image, Marvin File

Step 1. Deprotonation of the substrate α-amine, triggering CH bond cleavage in the next step.

Step 2. CH bond cleavage through the transfer of a hydride ion from the substrate to the flavin N5 atom.

Step 3. Non-enzymatic hydrolysis of intermediate iminoacid to α-keto acid and ammonia. Begins with the addition of water.

Step 4. Proton transfer.

Step 5. Elimination of ammonia.

Step 6. Deprotonation to form final product.

Products.

Introduction

A strong active site base abstracts the substrate α-proton with a pKa of ∼17. The resulting carbanion reacts with the flavin N5 atom, forming a covalent N5-flavin adduct. Cleavage of the substrate NH bond occurs stepwise with respect to CH bond cleavage, after formation of a covalent adduct with the flavin. Kinetic data suggesting NH and CH bond cleavage occur synchronously is not consistent with this mechanism.

Catalytic Residues Roles

UniProt PDB* (3nye)
Tyr53, Tyr249 Tyr1053(59)A, Tyr1249(255)A Stabilise transition state in PaDADH to facilitate the hydride transfer mechanism. transition state stabiliser
Glu87 Glu1087(93)A Stabilises D-arginine by engaging in an ionic interaction with the substrate guanido group. electrostatic stabiliser
His48 His1048(54)A His48 forms a hydrogen-bond network with the amino group of the substrate, separated by two water molecules. increase acidity
Ser45, Ala46 Ser1045(51)A, Ala1046(52)A In the FAD-binding site the side chains of S45 and A46 in loop L1, which do not interact directly with the substrate, adopt two alternate conformations depending on whether the ligand is present in the active site or not. steric role
*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

inferred reaction step, proton transfer, bimolecular nucleophilic addition, aromatic unimolecular elimination by the conjugate base, native state of cofactor is not regenerated, intermediate formation, hydrolysis, reaction occurs outside the enzyme, intramolecular elimination, intermediate terminated

References

  1. Gannavaram S et al. (2014), Biochemistry, 53, 6574-6583. Mechanistic and computational studies of the reductive half-reaction of tyrosine to phenylalanine active site variants of D-arginine dehydrogenase. DOI:10.1021/bi500917q. PMID:25243743.

Step 1. Deprotonation of the substrate α-amine.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ser1045(51)A steric role
Ala1046(52)A steric role
Tyr1053(59)A transition state stabiliser
Tyr1249(255)A transition state stabiliser
His1048(54)A increase acidity
Glu1087(93)A electrostatic stabiliser

Chemical Components

inferred reaction step, proton transfer

Step 2. Resulting carbanion reacts with the flavin N5 atom forming a covalent N5-flavin adduct.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles

Chemical Components

ingold: bimolecular nucleophilic addition

Step 3. Deprotonation of amine and cleavage of covalent N5-flavin adduct.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles

Chemical Components

ingold: aromatic unimolecular elimination by the conjugate base, native state of cofactor is not regenerated

Step 4. Non-enzymatic hydrolysis of intermediate iminoacid to α-keto acid and ammonia. Begins with the addition of water.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles

Chemical Components

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

Step 5. Proton transfer.

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Catalytic Residues Roles

Residue Roles

Chemical Components

intermediate formation, proton transfer, hydrolysis, reaction occurs outside the enzyme

Step 6. Elimination of ammonia.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles

Chemical Components

intermediate formation, ingold: intramolecular elimination, hydrolysis, reaction occurs outside the enzyme

Step 7. Deprotonation to form final product.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles

Chemical Components

intermediate terminated, proton transfer, hydrolysis, reaction occurs outside the enzyme
Download: Image, Marvin File

Step 1. Deprotonation of the substrate α-amine.

Step 2. Resulting carbanion reacts with the flavin N5 atom forming a covalent N5-flavin adduct.

Step 3. Deprotonation of amine and cleavage of covalent N5-flavin adduct.

Step 4. Non-enzymatic hydrolysis of intermediate iminoacid to α-keto acid and ammonia. Begins with the addition of water.

Step 5. Proton transfer.

Step 6. Elimination of ammonia.

Step 7. Deprotonation to form final product.

Products.

Introduction

A polar nucleophilic attack of the lone pair of electrons of the substrate α-amine at the flavin C4a atom may form a covalent C4a-flavin adduct. Cleavage of the substrate NH bond would occur stepwise with respect to CH bond cleavage, before formation of a covalent adduct with the flavin. Kinetic data suggesting NH and CH bond cleavage occur synchronously is not consistent with this mechanism.

Catalytic Residues Roles

UniProt PDB* (3nye)
Tyr53, Tyr249 Tyr1053(59)A, Tyr1249(255)A Stabilise transition state in PaDADH to facilitate the hydride transfer mechanism. transition state stabiliser
Glu87 Glu1087(93)A Stabilises D-arginine by engaging in an ionic interaction with the substrate guanido group. electrostatic stabiliser
His48 His1048(54)A His48 forms a hydrogen-bond network with the amino group of the substrate, separated by two water molecules. increase acidity
Ser45, Ala46 Ser1045(51)A, Ala1046(52)A In the FAD-binding site the side chains of S45 and A46 in loop L1, which do not interact directly with the substrate, adopt two alternate conformations depending on whether the ligand is present in the active site or not. steric role
*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, bimolecular nucleophilic addition, cofactor used, intermediate formation, native state of cofactor is not regenerated, unimolecular elimination by the conjugate base, reaction occurs outside the enzyme, hydrolysis, intramolecular elimination, intermediate terminated

References

  1. Gannavaram S et al. (2014), Biochemistry, 53, 6574-6583. Mechanistic and computational studies of the reductive half-reaction of tyrosine to phenylalanine active site variants of D-arginine dehydrogenase. DOI:10.1021/bi500917q. PMID:25243743.

Step 1. Deprotonation of the substrate α-amine.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu1087(93)A electrostatic stabiliser
Ser1045(51)A steric role
Ala1046(52)A steric role
Tyr1053(59)A transition state stabiliser
Tyr1249(255)A transition state stabiliser
His1048(54)A increase acidity

Chemical Components

proton transfer

Step 2. Polar nucleophilic attack of the lone pair of electrons of the substrate α-amine at the flavin C4a atom forming a covalent C4a-flavin adduct.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles

Chemical Components

ingold: bimolecular nucleophilic addition, cofactor used, intermediate formation

Step 3. Cleavage of covalent C4a-flavin adduct.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles

Chemical Components

native state of cofactor is not regenerated, ingold: unimolecular elimination by the conjugate base

Step 4. Non-enzymatic hydrolysis of intermediate iminoacid to α-keto acid and ammonia. Begins with the addition of water.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles

Chemical Components

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

Step 5. Proton transfer.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles

Chemical Components

reaction occurs outside the enzyme, hydrolysis, proton transfer, intermediate formation

Step 6. Elimination of ammonia.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles

Chemical Components

reaction occurs outside the enzyme, hydrolysis, ingold: intramolecular elimination, intermediate formation

Step 7. Deprotonation to form final product.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles

Chemical Components

reaction occurs outside the enzyme, hydrolysis, proton transfer, intermediate terminated
Download: Image, Marvin File

Step 1. Deprotonation of the substrate α-amine.

Step 2. Polar nucleophilic attack of the lone pair of electrons of the substrate α-amine at the flavin C4a atom forming a covalent C4a-flavin adduct.

Step 3. Cleavage of covalent C4a-flavin adduct.

Step 4. Non-enzymatic hydrolysis of intermediate iminoacid to α-keto acid and ammonia. Begins with the addition of water.

Step 5. Proton transfer.

Step 6. Elimination of ammonia.

Step 7. Deprotonation to form final product.

Products.

Contributors

Noa Marson, Antonio Ribeiro