2-oxopropyl-CoM reductase, carboxylating

 

Xanthobacter autotrophicus is able to grow on short chain aliphatic alkenes using a pathway whereby propylene can be converted into acetoacetate. The thioloxidoreductase/carboxylase enzyme described here catalyses the last step in this pathway, using 2-ketopropyl coenzyme M as a substrate and NADPH as the electron donor. The enzyme contains FAD as a cofactor and displays homology to the thiol oxidoreductase family including glutathione.

 

Reference Protein and Structure

Sequence
Q56839 UniProt (1.8.1.5) IPR004099 (Sequence Homologues) (PDB Homologues)
Biological species
Xanthobacter autotrophicus Py2 (Bacteria) Uniprot
PDB
1mok - NADPH DEPENDENT 2-KETOPROPYL COENZYME M OXIDOREDUCTASE/CARBOXYLASE (2.8 Å) PDBe PDBsum 1mok
Catalytic CATH Domains
3.30.390.30 CATHdb 3.50.50.60 CATHdb (see all for 1mok)
Cofactors
Fadh2(2-) (1)
Click To Show Structure

Enzyme Reaction (EC:1.8.1.5)

acetoacetate
CHEBI:13705ChEBI
+
coenzyme M(1-)
CHEBI:58319ChEBI
+
NADP(3-)
CHEBI:58349ChEBI
→
2-oxopropyl-CoM(1-)
CHEBI:57552ChEBI
+
carbon dioxide
CHEBI:16526ChEBI
+
NADPH(4-)
CHEBI:57783ChEBI
Alternative enzyme names: NADPH:2-(2-ketopropylthio)ethanesulfonate oxidoreductase/carboxylase, NADPH:2-ketopropyl-coenzyme M oxidoreductase/carboxylase, 2-KPCC,

Enzyme Mechanism

Introduction

The enzyme forms a disulfide bond from the residue cys 82 to coenzyme M, oxidising the substrate to form an enolactonate intermediate, stabilised by a water molecule activated by His 137 and Leu 78. Subsequent nucleophilic attack from cys 87 forms an intramolecular disulphide, which is in turn reduced by hydride transfer from FAD and ultimately from NADPH. The intramolecular disulphide is protected from hydrolysis by Phe 501, thus stabilising it. The enolactonate intermediate can act as a nucleophile, attacking Carbon dioxide to form acetoacetate.

Catalytic Residues Roles

UniProt PDB* (1mok)
Phe501 Phe501B Protects the intramolecular disulphide from hydrolysis, thus stabilising the intermediate. electrostatic stabiliser
His137 His137A Activates water through hydrogen bonding and acid-base interactions to allow the water molecule to stabilise the enolactonate intermediate. modifies pKa
Cys82 Cys82A Acts as a nucleophile to attack the sulphur atom of coenzyme M resulting in an intermolecular disulphide, causing the oxidation of the 2-keto-propyl moiety to the enolactonate transition state. covalent catalysis
Cys87 Cys87A Acts as secondary nucleophile to attack Cys 82 and form intramolecular disulphide, thus releasing Coenzyme M. Subsequent hydride transfer from FAD reoxidises this disulphide to give the catalytic form of the enzyme. covalent catalysis
Leu78 (main-C) Leu78A (main-C) Activates water through hydrogen bonding to allow it to act as a general acid base, stabilising the enolactone intermediate so that carboxylation can occur. modifies pKa, electrostatic stabiliser
*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. Nocek B et al. (2002), Biochemistry, 41, 12907-12913. Structural Basis for CO2Fixation by a Novel Member of the Disulfide Oxidoreductase Family of Enzymes, 2-Ketopropyl-Coenzyme M Oxidoreductase/Carboxylase†,‡. DOI:10.1021/bi026580p. PMID:12390015.
  2. Prussia GA et al. (2016), FEBS Lett, 590, 2991-2996. Substitution of a conserved catalytic dyad into 2-KPCC causes loss of carboxylation activity. DOI:10.1002/1873-3468.12325. PMID:27447465.
  3. Kofoed MA et al. (2011), J Bacteriol, 193, 4904-4913. Roles of the Redox-Active Disulfide and Histidine Residues Forming a Catalytic Dyad in Reactions Catalyzed by 2-Ketopropyl Coenzyme M Oxidoreductase/Carboxylase. DOI:10.1128/jb.05231-11. PMID:21764916.

Step 1.

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Catalytic Residues Roles

Residue Roles
Cys87A covalent catalysis
Cys82A covalent catalysis
Phe501B electrostatic stabiliser
Leu78A (main-C) electrostatic stabiliser, modifies pKa
His137A modifies pKa

Chemical Components

Step 1.

Contributors

Hannah Gilbert, Peter Sarkies, Gemma L. Holliday