Dimethylglycine oxidase (tetrahydrofolate utilising)

 

Catalyses the oxidative demethylation of N,N-dimethylglycine to yield sarcosine, 5,10-methylenetetrahydrofolate and hydrogen peroxide. The oxidation of dimethylglycine is coupled to the synthesis of 5,10-methylenetetrahydrofolate through an unusual substrate channelling mechanism. This channelling occurs by non-biased diffusion of the iminium intermediate through a large solvent cavity connecting active site 1 (N-terminus) and active site 2 (C-terminus). The synthesis of 5,10-methylenetetrahydrofolate (at active site 2) prevents the accumulation of formaldehyde (not shown), formed by hydrolysis of the iminium intermediate product (at active site 1).

 

Reference Protein and Structure

Sequence
Q9AGP8 UniProt (1.5.3.10) IPR006076 (Sequence Homologues) (PDB Homologues)
Biological species
Arthrobacter globiformis (Bacteria) Uniprot
PDB
1pj5 - Crystal structure of dimethylglycine oxidase of Arthrobacter globiformis in complex with acetate (1.61 Å) PDBe PDBsum 1pj5
Catalytic CATH Domains
3.30.9.10 CATHdb 3.30.70.1400 CATHdb (see all for 1pj5)
Cofactors
Fadh2(2-) (1)
Click To Show Structure

Enzyme Reaction (EC:1.5.3.-)

dioxygen
CHEBI:15379ChEBI
+
N,N-dimethylglycine zwitterion
CHEBI:58251ChEBI
+
(6S)-5,6,7,8-tetrahydrofolate(2-)
CHEBI:57453ChEBI
→
hydron
CHEBI:15378ChEBI
+
hydrogen peroxide
CHEBI:16240ChEBI
+
(6R)-5,10-methylenetetrahydrofolate(2-)
CHEBI:15636ChEBI
+
sarcosinate
CHEBI:46915ChEBI

Enzyme Mechanism

Introduction

The initial deprotonation of the substrate amine by the His225–Tyr259 catalytic dyad is followed by the attack of the substrate on the FAD C4 position. A proton is abstracted from the covalent intermediate methyl group by the N5, with concomitant reduction of the isoalloxazine ring and release of the dimethylglycine iminium ion. The imminium ion is then transferred to the second active site. Here, the bound THF attacks the iminium ion via the nucleophilic N10 group with concomitant proton abstraction by Asp552. The next step involves the attack by the N5 group on the covalent intermediate with concomitant deprotonation of the N5 by the nascent sarcosine. An alternative possibility is that the N5 position attacks first and donates a proton to the covalent intermediate, followed by the nucleophilic attack of the N10 group with release of sarcosine.

Catalytic Residues Roles

UniProt PDB* (1pj5)
His225, Tyr259 His225A, Tyr259A Forms a Tyr-His dyad that is responsible for the initial deprotonation that initiates the reaction at active site 1. proton shuttle (general acid/base)
Asp552 Asp552A Acts as the general acid/base in the second half reaction performed by this enzyme in active site 2. proton shuttle (general acid/base)
*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

References

  1. Leys D et al. (2003), EMBO J, 22, 4038-4048. Channelling and formation of 'active' formaldehyde in dimethylglycine oxidase. DOI:10.1093/emboj/cdg395. PMID:12912903.
  2. Tralau T et al. (2009), J Biol Chem, 284, 17826-17834. An internal reaction chamber in dimethylglycine oxidase provides efficient protection from exposure to toxic formaldehyde. DOI:10.1074/jbc.M109.006262. PMID:19369258.

Step 1.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His225A proton shuttle (general acid/base)
Tyr259A proton shuttle (general acid/base)
Asp552A proton shuttle (general acid/base)

Chemical Components

Step 1.

Contributors

Alex Gutteridge, Craig Porter, Gemma L. Holliday