Protein-disulfide reductase

 

Disulfide bond (Dsb) proteins are required to form disulphide bonds which enable the folding of secreted proteins within the periplasm of Escherichia coli. DsbA catalyses the oxidation of a pair of cysteine sulfhydryl groups, forming disulphide bonds. Electrons generated from the oxidation are transferred from DsbA to DsbB then into the electron transport chain. DsbC, DsbD, DsbE and DsbG repair improperly formed disulfides.

The N terminal region of the thioloxidoreductase enzyme DsbD from E. coli is able to reduce the disulphide bond in DsbC and thus participates in the pathway whereby electrons from NADPH are used to break incorrectly formed disulphides in proteins in the periplasm. It displays a unique Ig fold, but still shows homology with the thioloxidoreductase family.

 

Reference Protein and Structure

Sequence
P36655 UniProt (1.8.1.8) IPR022910 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
1l6p - N-terminal of DsbD (residues 20-144) from E. coli. (1.65 Å) PDBe PDBsum 1l6p
Catalytic CATH Domains
2.60.40.1250 CATHdb (see all for 1l6p)
Click To Show Structure

Enzyme Reaction (EC:1.8.1.8)

L-cystine residue
CHEBI:50058ChEBI
+
hydron
CHEBI:15378ChEBI
+
NADH(2-)
CHEBI:57945ChEBI
→
L-cysteine residue
CHEBI:29950ChEBI
+
NAD(1-)
CHEBI:57540ChEBI
Alternative enzyme names: NAD(P)H(2):protein-disulfide oxidoreductase, Disulfide reductase, Insulin-glutathione transhydrogenase, Protein disulfide reductase, NAD(P)H:protein-disulfide oxidoreductase,

Enzyme Mechanism

Introduction

Cys 109 is the primary nucleophile that attacks the intramolecular disulphide to form a disulphide between DsbD and DsbC. Cys 103 then acts as a secondary nucleophile to reduce this disulphide, activated by a Asp 68-Tyr 42 diad which acts as a proton relay system. This results in the reduced form intermediate, stabilised by contacts with Tyr 71 and Phe 70

Catalytic Residues Roles

UniProt PDB* (1l6p)
Asp87 Asp68A Activates Tyr 42 to allow it to act as a general acid-base, thus is part of an Asp-Tyr diad which functions as a proton relay system to activate Cys 103. proton acceptor, electrostatic stabiliser, proton donor
Phe89 Phe70A Makes hydrophobic contact with intramolecular disulphide of reduced form to stabilise it by preventing nucleophilic attack by a water molecule. electrostatic stabiliser
Tyr90 Tyr71A Acts to shield the reduced form from nucleophilic attack, thus stabilising it. electrostatic stabiliser
Tyr61 Tyr42A Acts to deprotonate Cys 103 thus allowing it to form the intramolecular disulphide with Cys 109 that characterises the reduced form of the enzyme. proton relay, proton acceptor, proton donor
Cys122 Cys103A Acts as nucleophile to reduce disulphide bond between DsbC and DsbD, forming the reduced intermediate. hydride acceptor, electrofuge, nucleophile, proton donor
Cys128 Cys109A Acts as primary nucleophile to break the intramolecular disulphide bond in DsbC which leads to the reduced form tof the enzyme with an intramolecular disulphide bond between Cys 109 and 103. covalently attached, nucleofuge, nucleophile, electrofuge, electrophile
*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, intermediate formation, enzyme-substrate complex formation, proton transfer, overall product formed, enzyme-substrate complex cleavage, inferred reaction step, native state of enzyme regenerated

References

  1. Goulding CW et al. (2002), Biochemistry, 41, 6920-6927. Thiol−Disulfide Exchange in an Immunoglobulin-like Fold:  Structure of the N-Terminal Domain of DsbD†,‡. DOI:10.1021/bi016038l. PMID:12033924.
  2. Nakamoto H et al. (2004), Biochim Biophys Acta, 1694, 111-119. Catalysis of disulfide bond formation and isomerization in the Escherichia coli periplasm. DOI:10.1016/j.bbamcr.2004.02.012. PMID:15546661.
  3. Haebel PW et al. (2002), EMBO J, 21, 4774-4784. The disulfide bond isomerase DsbC is activated by an immunoglobulin-fold thiol oxidoreductase: crystal structure of the DsbC-DsbDalpha complex. DOI:10.1093/emboj/cdf489. PMID:12234918.
  4. Nikol'skaia II et al. (1975), Biokhimiia, 40, 875-877. Determination of the nature of a bond between glucose and 5'-hydrocymethylcytosine in DDVI phage DNA. PMID:1203394.

Step 1. The thiolate form of Cys109 performs a nucleophilic attack on the oxidized target protein DsbC, forming and intermolecular disulfide bond.

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Catalytic Residues Roles

Residue Roles
Asp68A electrostatic stabiliser
Tyr71A electrostatic stabiliser
Phe70A electrostatic stabiliser
Cys109A covalently attached, nucleophile

Chemical Components

ingold: bimolecular nucleophilic substitution, intermediate formation, enzyme-substrate complex formation

Step 2. Asp68 acts as a general acid to remove a proton from Tyr42 which then activates Cys103. This triggers the nucleophilic attack of Cys103 on the mixed disulfide bridge.

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Catalytic Residues Roles

Residue Roles
Cys103A nucleophile
Cys109A electrofuge
Tyr42A proton acceptor
Asp68A proton acceptor
Tyr42A proton donor
Cys103A proton donor
Cys109A electrophile
Tyr42A proton relay

Chemical Components

proton transfer, ingold: bimolecular nucleophilic substitution, overall product formed, enzyme-substrate complex cleavage

Step 3. In an inferred step the L-cysteine residue is protonated and the product is formed.

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Catalytic Residues Roles

Residue Roles

Chemical Components

proton transfer, overall product formed, inferred reaction step

Step 4. DsbD receives electrons from the cytoplasmic thioredoxin system. In an inferred reaction step the Cys103 - Cys109 disulfide bridge is reduced by electrons from this domain to restore the native state of the enzyme. This is illustrated using NADPH which is the original electron donor, however, NADPH is not present in this domain.

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Catalytic Residues Roles

Residue Roles
Cys103A electrofuge
Cys109A nucleofuge
Cys103A hydride acceptor

Chemical Components

inferred reaction step, native state of enzyme regenerated

Step 5. In an inferred reaction step Asp 68 is protonated to regenerate the native state of the enzyme.

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Catalytic Residues Roles

Residue Roles
Asp68A proton donor

Chemical Components

proton transfer, native state of enzyme regenerated, inferred reaction step
Download: Image, Marvin File

Step 1. The thiolate form of Cys109 performs a nucleophilic attack on the oxidized target protein DsbC, forming and intermolecular disulfide bond.

Step 2. Asp68 acts as a general acid to remove a proton from Tyr42 which then activates Cys103. This triggers the nucleophilic attack of Cys103 on the mixed disulfide bridge.

Step 3. In an inferred step the L-cysteine residue is protonated and the product is formed.

Step 4. DsbD receives electrons from the cytoplasmic thioredoxin system. In an inferred reaction step the Cys103 - Cys109 disulfide bridge is reduced by electrons from this domain to restore the native state of the enzyme. This is illustrated using NADPH which is the original electron donor, however, NADPH is not present in this domain.

Step 5. In an inferred reaction step Asp 68 is protonated to regenerate the native state of the enzyme.

Products.

Contributors

Peter Sarkies, Gemma L. Holliday, Amelia Brasnett