Protein-methionine-S-oxide reductase (MsrB)

 

The oxidation of methionine residues to methionine sulfoxide is implicated in ageing processes; the reduction of methionine sulfoxide protein residues is similarly implicated in antioxidant repair activity, but also the virulence of pathogenic organisms, such as Neisseria gonorrhoeae which secretes pilB. The modular pilB contains three domains; one is a methionine sulfoxide reductase (MsrA) which reduces S-methionine sulfoxide (S-chiral at sulphur), and another an MsrB (this entry) which reduces R-methionine sulfoxide. The two domains have no sequence homology but use the same mechanism - an example of the fusion of two convergently evolved enzymes.

 

Reference Protein and Structure

Sequence
P14930 UniProt (1.8.4.11, 1.8.4.12) IPR002579 (Sequence Homologues) (PDB Homologues)
Biological species
Neisseria gonorrhoeae (Bacteria) Uniprot
PDB
1l1d - Crystal structure of the C-terminal methionine sulfoxide reductase domain (MsrB) of N. gonorrhoeae pilB (1.85 Å) PDBe PDBsum 1l1d
Catalytic CATH Domains
2.170.150.20 CATHdb (see all for 1l1d)
Click To Show Structure

Enzyme Reaction (EC:1.8.4.11)

L-methionine (S)-S-oxide residue
CHEBI:44120ChEBI
+
L-cysteine residue
CHEBI:29950ChEBI
→
L-methionine residue
CHEBI:16044ChEBI
+
water
CHEBI:15377ChEBI
+
L-cystine residue
CHEBI:50058ChEBI
Alternative enzyme names: MsrA, Methionine sulfoxide reductase, Methionine sulphoxide reductase A, Methionine S-oxide reductase, Methionine S-oxide reductase (S-form oxidizing), Methionine sulfoxide reductase A, Peptide Met(O) reductase, Peptide methionine sulfoxide reductase,

Enzyme Mechanism

Introduction

Cys 495 exists as a thiolate due to the conserved interactions with Arg 493 and Asp 484. Attack of Cys 495 on the sulphur of methionine sulfoxide leads to a 1,3-sigmatropic rearrangement involving the sulphur of Cys 495, and the S=O atoms of the substrate. The trigonal bipyramid transition state (one equatorial position occupied by the substrate sulphur lone pair) is stabilised by hydrogen bonding to His 480 and a fixed water molecule. The transition state collapses to yield methionine and a sulfenic acid intermediate, with Cys 495 hydroxylated as Cys 495-SOH . Cys 440 is also a thiolate and forms a disulfide bond with Cys 495, releasing the hydroxyl group as water. The disulfide bond is reduced by thioredoxin to regenerate the free thiolates.

Catalytic Residues Roles

UniProt PDB* (1l1d)
Cys440 Cys440(70)B Cys 440 attacks the sulfenic acid intermediate of Cys 495, forming a disulfide bond and displacing the hydroxyl as water. nucleofuge, nucleophile, proton acceptor
His480 His480(110)B His 480 is the oxyanion hole, stabilising the trigonal bipyramid transition state via hydrogen bonding to the negative charge on the substrate oxygen. proton acceptor, electrostatic stabiliser, proton donor
Asp484 Asp484(114)B Asp 484 hydrogen bonds to Arg 493, making Arg 493 a better stabiliser of the thiolate charge. electrostatic stabiliser
Arg493 Arg493(123)B Arg 493 hydrogen bonds to the Cys 495 thiolate, stabilising the negative charge (i.e. lowers the pKa of the neutral form). electrostatic stabiliser
Cys495 Cys495(125)B The Cys 495 thiolate attacks the sulphur of the methionine sulfoxide residue, leading to a 1,3-sigmatropic rearrangement with the sulfoxide group. The oxygen of the sulfoxide is therefore transferred to Cys 495 to give a sulfenic acid intermediate. 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 addition, proton transfer, overall reactant used, sigmatropic rearrangement, overall product formed, bimolecular nucleophilic substitution, enzyme-substrate complex formation, enzyme-substrate complex cleavage

References

  1. Olry A et al. (2004), Biochemistry, 43, 11616-11622. Kinetic Characterization of the Catalytic Mechanism of Methionine Sulfoxide Reductase B fromNeisseria meningitidis†. DOI:10.1021/bi049306z. PMID:15350148.
  2. Olry A et al. (2002), J Biol Chem, 277, 12016-12022. Characterization of the Methionine Sulfoxide Reductase Activities of PILB, a Probable Virulence Factor fromNeisseria meningitidis. DOI:10.1074/jbc.m112350200. PMID:11812798.
  3. Lowther WT et al. (2002), Nat Struct Biol, 9, 348-352. The mirrored methionine sulfoxide reductases of Neisseria gonorrhoeae pilB. DOI:10.1038/nsb783. PMID:11938352.

Step 1. Catalysis begins with nucleophilic attack of Cys495 on the sulfoxide group of the reactant. The developing oxyanion abstracts a proton from His480.

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Catalytic Residues Roles

Residue Roles
His480(110)B electrostatic stabiliser
Arg493(123)B electrostatic stabiliser
Cys495(125)B covalently attached
Asp484(114)B electrostatic stabiliser
Cys495(125)B nucleophile
His480(110)B proton donor

Chemical Components

ingold: bimolecular nucleophilic addition, proton transfer, overall reactant used

Step 2. There is a 1,3 sigmatropic rearrangement leading to the formation of a sulfenic acid intermediate and the release of the methionine product.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys495(125)B covalently attached
His480(110)B electrostatic stabiliser
Asp484(114)B electrostatic stabiliser
Arg493(123)B electrostatic stabiliser
Cys495(125)B electrophile, electrofuge

Chemical Components

sigmatropic rearrangement, overall product formed

Step 3. Cys495 is subject to nucleophilic attack by Cys440, releasing the hydroxyl group which accepts a proton from a donor to form water.

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Catalytic Residues Roles

Residue Roles
His480(110)B electrostatic stabiliser
Asp484(114)B electrostatic stabiliser
Arg493(123)B electrostatic stabiliser
Cys495(125)B electrofuge, electrophile
Cys440(70)B nucleophile

Chemical Components

ingold: bimolecular nucleophilic substitution

Step 4. In a series of inferred steps the newly formed disulfide bond between Cys440 and Cys495 is reduced by thioredoxin. This begins with nucleophilic attack on the disulfide bond by a thioredoxin thiol.

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Catalytic Residues Roles

Residue Roles
Cys495(125)B electrophile
Cys440(70)B nucleofuge
Cys495(125)B electrofuge
Cys440(70)B proton acceptor

Chemical Components

ingold: bimolecular nucleophilic substitution, proton transfer, enzyme-substrate complex formation

Step 5. The second thioredoxin residue initiates nucleophilic attack on the thioredoxin Cys495 disulfide bond, eliminating Cys495 and forming a new disulfide bond.

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Catalytic Residues Roles

Residue Roles
Cys495(125)B nucleofuge

Chemical Components

enzyme-substrate complex cleavage, ingold: bimolecular nucleophilic substitution

Step 6. In an inferred step His480 is reprotonated to regenerate the active site.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His480(110)B proton acceptor

Chemical Components

proton transfer
Download: Image, Marvin File

Step 1. Catalysis begins with nucleophilic attack of Cys495 on the sulfoxide group of the reactant. The developing oxyanion abstracts a proton from His480.

Step 2. There is a 1,3 sigmatropic rearrangement leading to the formation of a sulfenic acid intermediate and the release of the methionine product.

Step 3. Cys495 is subject to nucleophilic attack by Cys440, releasing the hydroxyl group which accepts a proton from a donor to form water.

Step 4. In a series of inferred steps the newly formed disulfide bond between Cys440 and Cys495 is reduced by thioredoxin. This begins with nucleophilic attack on the disulfide bond by a thioredoxin thiol.

Step 5. The second thioredoxin residue initiates nucleophilic attack on the thioredoxin Cys495 disulfide bond, eliminating Cys495 and forming a new disulfide bond.

Step 6. In an inferred step His480 is reprotonated to regenerate the active site.

Products.

Introduction

In a computational study a mechanism involving a sulfonium ion intermediate was proposed. The reaction begins with nucleophilic attack of Cys495 on the sulfoxide of the substrate, within concomitant proton transfer from His480. Next, protonation of the sulfurane oxygen by His477 occurs. Finally there is direct nucleophilic attack of Cys440 on the Cys495-substrate bond, releasing water and methionine. The Cys440-Cys495 disulfide bond is reduced by thioredoxin to regenerate the native state of the enzyme.

Catalytic Residues Roles

UniProt PDB* (1l1d)
His477 His477(107)B His477 is the most likely proton source for protonation of the oxygen in the sulfurane intermediate. proton acceptor, proton donor
Cys440 Cys440(70)B Cys 440 attacks the sulfonium ion intermediate of Cys 495, forming a disulfide bond and displacing the hydroxyl as water. nucleofuge, nucleophile, proton acceptor
His480 His480(110)B His 480 protonates and forming oxyanion on the sulfurane intermediate after nucleophilic attack of the substrate by Cys 495. proton acceptor, electrostatic stabiliser, proton donor
Asp484 Asp484(114)B Asp 484 hydrogen bonds to Arg 493, making Arg 493 a better stabiliser of the thiolate charge. electrostatic stabiliser
Arg493 Arg493(123)B Arg 493 hydrogen bonds to the Cys 495 thiolate, stabilising the negative charge (i.e. lowers the pKa of the neutral form). electrostatic stabiliser
Cys495 Cys495(125)B The Cys495 thiolate attacks the sulphur of the methionine sulfoxide residue, forming an enzyme-substrate complex. The disulfide bond is cleaved after nucleophilic attack from Cys440. electrophile, electrofuge, 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 addition, enzyme-substrate complex formation, proton transfer, bimolecular nucleophilic substitution, overall product formed, enzyme-substrate complex cleavage, native state of enzyme regenerated

References

  1. Robinet JJ et al. (2011), J Phys Chem B, 115, 9202-9212. A sulfonium cation intermediate in the mechanism of methionine sulfoxide reductase B: a DFT study. DOI:10.1021/jp111681e. PMID:21721538.
  2. Olry A et al. (2004), Biochemistry, 43, 11616-11622. Kinetic Characterization of the Catalytic Mechanism of Methionine Sulfoxide Reductase B fromNeisseria meningitidis†. DOI:10.1021/bi049306z. PMID:15350148.
  3. Olry A et al. (2002), J Biol Chem, 277, 12016-12022. Characterization of the Methionine Sulfoxide Reductase Activities of PILB, a Probable Virulence Factor fromNeisseria meningitidis. DOI:10.1074/jbc.m112350200. PMID:11812798.

Step 1. Cys495 initiates nucleophilic attack on the sulfoxide sulfur of the reactant. The developing oxyanion is protonated by His480.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp484(114)B electrostatic stabiliser
Arg493(123)B electrostatic stabiliser
His480(110)B proton donor
Cys495(125)B nucleophile

Chemical Components

ingold: bimolecular nucleophilic addition, enzyme-substrate complex formation, proton transfer

Step 2. The sulfurane intermediate is protonated to form a sulfonium ion intermediate. The proton source is most likely to be His477.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His480(110)B electrostatic stabiliser
Asp484(114)B electrostatic stabiliser
Arg493(123)B electrostatic stabiliser
His477(107)B proton donor

Chemical Components

proton transfer

Step 3. There is direct nucleophilic attack of Cys440 on Cys495, forming methionine and releasing water in the process.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys440(70)B nucleophile
Cys495(125)B electrofuge, electrophile

Chemical Components

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

Step 4. In a series of inferred steps the newly formed disulfide bond between Cys440 and Cys495 is reduced by thioredoxin. This begins with nucleophilic attack on the disulfide bond by a thioredoxin thiol.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys495(125)B electrofuge
Cys440(70)B proton acceptor
Cys495(125)B electrophile
Cys440(70)B nucleofuge

Chemical Components

ingold: bimolecular nucleophilic substitution, proton transfer, enzyme-substrate complex formation

Step 5. The second thioredoxin residue initiates nucleophilic attack on the thioredoxin Cys495 disulfide bond, eliminating Cys495 and forming a new disulfide bond.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys495(125)B nucleofuge

Chemical Components

ingold: bimolecular nucleophilic substitution, enzyme-substrate complex cleavage, overall product formed, native state of enzyme regenerated

Step 6. In an inferred step His480 and His477 are both reprotonated to regenerate the native state of the enzyme.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His480(110)B proton acceptor
His477(107)B proton acceptor

Chemical Components

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

Step 1. Cys495 initiates nucleophilic attack on the sulfoxide sulfur of the reactant. The developing oxyanion is protonated by His480.

Step 2. The sulfurane intermediate is protonated to form a sulfonium ion intermediate. The proton source is most likely to be His477.

Step 3. There is direct nucleophilic attack of Cys440 on Cys495, forming methionine and releasing water in the process.

Step 4. In a series of inferred steps the newly formed disulfide bond between Cys440 and Cys495 is reduced by thioredoxin. This begins with nucleophilic attack on the disulfide bond by a thioredoxin thiol.

Step 5. The second thioredoxin residue initiates nucleophilic attack on the thioredoxin Cys495 disulfide bond, eliminating Cys495 and forming a new disulfide bond.

Step 6. In an inferred step His480 and His477 are both reprotonated to regenerate the native state of the enzyme.

Products.

Contributors

Jonathan T. W. Ng, Gemma L. Holliday, Amelia Brasnett