Protein disulfide oxidoreductase

 

DsbB isolated from Escherichia coli is able to create disulfide bonds de novo. It forms a complex with DsbA and overall there is a net formation of a disulfide bridge in DsbA and a quinone cofactor is reduced in DsbB. The quinone can either be ubiquinone (under aerobic conditions) or menaquinone (under anaerobic conditions). DsbA is a periplasmic dithiol oxidase and it is the primary disulfide bond donor in the periplasm. The disulfide bond to be produced in DsbA is between Cys30 and Cys33. Two disulfide bonds exist in DsbB in the resting state, Cys41-Cys44 and Cys104-Cys130; they are involved in the catalytic pathway.

 

Reference Protein and Structure

Sequences
P0AEG4 UniProt
P0A6M2 UniProt IPR022920 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
2hi7 - Crystal structure of DsbA-DsbB-ubiquinone complex (3.7 Å) PDBe PDBsum 2hi7
Catalytic CATH Domains
1.20.1550.10 CATHdb (see all for 2hi7)
Cofactors
Ubiquinone-1 (1)
Click To Show Structure

Enzyme Reaction (EC:1.8.4.-)

ubiquinones
CHEBI:16389ChEBI
+
L-cysteine residue
CHEBI:29950ChEBI
→
ubiquinol
CHEBI:17976ChEBI
+
L-cystine residue
CHEBI:50058ChEBI

Enzyme Mechanism

Introduction

DsbB contains the Cys41-Cys44 and Cys104-Cys130 disulfide bonds in the resting state. In the DsbA-DsbB complex, DsbA causes a conformational change which separates Cys104 and Cys130. The reaction begins witgh nucleophilic attack by the DsbA Cys33 thiolate on the intermolecular Cys104-Cys30 disulfide bond leading to a Cys30-Cys33 intramolecular disulfide bond and a Cys104 thiolate. The hemi-oxidised DsbB then rearranges so that the Cys41-Cys44 disulfide bond is reduced and Cys104 and Cys130 are oxidised to form a bond. The Cys44 thiolate forms a charge transfer complex with the quinone, stabilised by Arg48, and then forms an adduct through the C2 of the quinone. The negative charge is delocalised around the O2 atom and is also stabilised by Arg48. The Cys41 thiolate then acts as a nucleophile and attacks Cys44, breaking the S-C2 bond and creating a disulfide bridge and a reduced quinone molecule.
The above refers to the rapid pathway, which predominates. There is a minor pathway called the slow pathway. The two diverge after the formation of the intermolecular disulfide bond. In the slow pathway there is nucleophilic attack on the Cys41-Cys44 disulfide bond by the Cys130 thiolate, leading to a Cys41-Cys130 disulfide bond and a Cys44 thiolate. The exact mechanism by which the final disulfide bonds are generated and the quinone is reduced is unknown.

Catalytic Residues Roles

UniProt PDB* (2hi7)
Cys130 SerNone(130)B In the resting state of DsbB there is a Cys130-Cys104 disulfide bond. The Cys104–Cys130 pair is involved directly in the disulfide exchange with DsbA electrofuge, electrophile, nucleophile
Cys41 Cys41B The Cys41-Cys44 disulfide bond is reduced in the hemi-oxidised DsbB. The Cys41 thiolate attacks the Cys44-quinone adduct to reform the Cys41-Cys44 disulfide bond. electrofuge, electrophile, nucleophile, nucleofuge
Cys44 Cys44B The Cys41-Cys44 disulfide bond is reduced in the hemi-oxidised DsbB. The Cys44 thiolate forms a charge transfer complex with the quinone and then an adduct. The sulfur acts as the electrophile in the nucleophilic substitution by Cys41, thus reforming the disulfide bond. electrophile, electrofuge, nucleofuge, nucleophile
Arg48 Arg48B Arg48 stabilises the charge transfer complex between the Cys44 thiolate and the quinone. It also stabilises the adduct formed between the two. electrostatic stabiliser
Cys104 Cys104B The Cys104-Cys130 bond is cleaved by nucleophilic attack by the Cys30 thiolate, forming a Cys104-Cys30 bond. This bond is then cleaved after nucleophilic attack by the Cys33 thiolate, leaving Cys104 as a thiolate. The rearrangement of the hemi-oxidised DsbB allows the Cys104-Cys130 bond to reform when the Cys41-Cys44 bond is reduced. covalently attached, nucleofuge, nucleophile
*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 nucleophilic substitution, overall product formed, bimolecular nucleophilic addition, michael addition, overall reactant used, intermediate formation, enzyme-substrate complex formation, proton transfer, native state of enzyme regenerated, enzyme-substrate complex cleavage

References

  1. Inaba K et al. (2006), Cell, 127, 789-801. Crystal Structure of the DsbB-DsbA Complex Reveals a Mechanism of Disulfide Bond Generation. DOI:10.1016/j.cell.2006.10.034. PMID:17110337.
  2. Inaba K et al. (2009), EMBO J, 28, 779-791. Dynamic nature of disulphide bond formation catalysts revealed by crystal structures of DsbB. DOI:10.1038/emboj.2009.21. PMID:19214188.
  3. Inaba K et al. (2008), Biochim Biophys Acta, 1783, 520-529. Structure and mechanisms of the DsbB–DsbA disulfide bond generation machine. DOI:10.1016/j.bbamcr.2007.11.006. PMID:18082634.
  4. Inaba K et al. (2006), Proc Natl Acad Sci U S A, 103, 287-292. Critical role of a thiolate-quinone charge transfer complex and its adduct form in de novo disulfide bond generation by DsbB. DOI:10.1073/pnas.0507570103. PMID:16384917.
  5. Kishigami S et al. (1996), Genes Cells, 1, 201-208. Roles of cysteine residues of DsbB in its activity to reoxidize DsbA, the protein disulphide bond catalyst ofEscherichia coli. DOI:10.1046/j.1365-2443.1996.d01-233.x. PMID:9140064.

Step 1. DsbB contains the Cys41-Cys44 and Cys104-Cys130 disulfide bonds in the resting state. In the DsbA-DsbB complex, DsbA causes a conformational change which separates Cys104 and Cys130. The rapid pathway begins when Cys33 of DsbA attacks the intermolecular Cys104 (DsbB) - Cys30 (DbsA) disulfide bond. This forms the Cys30 - Cys33 disulfide product in DsbA.

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Catalytic Residues Roles

Residue Roles
Cys104B covalently attached
Cys104B nucleofuge

Chemical Components

ingold: bimolecular nucleophilic substitution, overall product formed

Step 2. Cys130 then initiates nucleophilic attack the Cys44-Cys41 disulfide bond. This is the first step in a dithiol disulfide exchange between the active site residues.

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Catalytic Residues Roles

Residue Roles
Cys41B electrofuge
SerNone(130)B nucleophile
Cys41B electrophile
Cys44B nucleofuge

Chemical Components

ingold: bimolecular nucleophilic substitution

Step 3. There is nucleophilic attack of Cys104 on the Cys130-Cys41 disulfide bond. In comparison to the original resting state of DsbB, the Cys41-Cys44 disulfide bond has been reduced by two electrons.

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Catalytic Residues Roles

Residue Roles
SerNone(130)B electrofuge, electrophile
Cys41B nucleofuge
Cys104B nucleophile

Chemical Components

ingold: bimolecular nucleophilic substitution

Step 4. Cys44 forms a charge transfer complex with ubiquinone before initiating nucleophilic attack and forming an adduct. Arg48 stabilises Cys44 and the adduct formed.

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Catalytic Residues Roles

Residue Roles
Arg48B electrostatic stabiliser
Cys44B nucleophile

Chemical Components

ingold: bimolecular nucleophilic addition, michael addition, overall reactant used, intermediate formation, enzyme-substrate complex formation, proton transfer

Step 5. The S-C covalent linkage in the adduct is attacked by Cys41, reforming the Cys41-Cys44 disulfide bond and resulting in a quinol. The quinol is subsequently protonated to complete the reaction.

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Catalytic Residues Roles

Residue Roles
Arg48B electrostatic stabiliser
Cys44B electrophile, electrofuge
Cys41B nucleophile

Chemical Components

ingold: bimolecular nucleophilic substitution, native state of enzyme regenerated, overall product formed, enzyme-substrate complex cleavage
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Step 1. DsbB contains the Cys41-Cys44 and Cys104-Cys130 disulfide bonds in the resting state. In the DsbA-DsbB complex, DsbA causes a conformational change which separates Cys104 and Cys130. The rapid pathway begins when Cys33 of DsbA attacks the intermolecular Cys104 (DsbB) - Cys30 (DbsA) disulfide bond. This forms the Cys30 - Cys33 disulfide product in DsbA.

Step 2. Cys130 then initiates nucleophilic attack the Cys44-Cys41 disulfide bond. This is the first step in a dithiol disulfide exchange between the active site residues.

Step 3. There is nucleophilic attack of Cys104 on the Cys130-Cys41 disulfide bond. In comparison to the original resting state of DsbB, the Cys41-Cys44 disulfide bond has been reduced by two electrons.

Step 4. Cys44 forms a charge transfer complex with ubiquinone before initiating nucleophilic attack and forming an adduct. Arg48 stabilises Cys44 and the adduct formed.

Step 5. The S-C covalent linkage in the adduct is attacked by Cys41, reforming the Cys41-Cys44 disulfide bond and resulting in a quinol. The quinol is subsequently protonated to complete the reaction.

Products.

Contributors

Gemma L. Holliday, Amelia Brasnett