6-deoxyerythronolide-B synthase

 

Thioesterase domain of 6-deoxyerythronolide B synthase, a polyketide synthase, catalyses the cyclisation and release of 6-deoxyerythronolide B via lactonisation. 6-deoxyerythronolide B is the macrocyclic core of the antibiotic erythromycin

 

Reference Protein and Structure

Sequence
Q03133 UniProt (2.3.1.94) IPR016039 (Sequence Homologues) (PDB Homologues)
Biological species
Saccharopolyspora erythraea (Bacteria) Uniprot
PDB
1kez - Crystal Structure of the Macrocycle-forming Thioesterase Domain of Erythromycin Polyketide Synthase (DEBS TE) (2.8 Å) PDBe PDBsum 1kez
Catalytic CATH Domains
3.40.50.1820 CATHdb (see all for 1kez)
Click To Show Structure

Enzyme Reaction (EC:2.3.1.94)

6-deoxyerythronolide B linear precursor
CHEBI:X00672X00672
→
coenzyme A(4-)
CHEBI:57287ChEBI
+
6-deoxyerythronolide B
CHEBI:16089ChEBI
Alternative enzyme names: Erythronolide-condensing enzyme, Malonyl-CoA:propionyl-CoA malonyltransferase (cyclizing), Malonyl-CoA:propanoyl-CoA malonyltransferase (cyclizing), Erythronolide synthase, Deoxyerythronolide B synthase, 6-deoxyerythronolide B synthase, DEBS,

Enzyme Mechanism

Introduction

The catalytic triad consists of Asp169, His259 and Ser142. His259, stabilised by Asp-169, acts as a base that deprotonates Ser-142, as it attacks the ACP6-bound thioester substrate, to yield an acyl-O-serine intermediate. The NH of Ala-143 may serve as the oxyanion stabilising residue. The enzyme-bound intermediate is subsequently attacked by a hydroxyl group on the polyketide chain.

Catalytic Residues Roles

UniProt PDB* (1kez)
His3148 His259A It deprotonates Ser 142 to allow its nucleophilic attack of thioester substrate to yield an acyl-O-serine intermediate. proton acceptor, proton donor
Asp3058 Asp169A It alters the pKa of His 259 and stabilise it for the deprotonation of Ser 142 modifies pKa, electrostatic stabiliser
Ala3032 (main-N) Ala143A (main-N) Its backbone NH lines the oxyanion hole and acts as a hydrogen bond donor to the C1 carbonyl electrostatic stabiliser
Ser3031 Ser142A It nucleophilically attacks the thioester substrate upon deprotonation by His 259. covalently attached, nucleofuge, nucleophile, proton acceptor, proton donor
*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

proton transfer, bimolecular nucleophilic addition, enzyme-substrate complex formation, intermediate formation, overall reactant used, unimolecular elimination by the conjugate base, intermediate collapse, overall product formed, enzyme-substrate complex cleavage, intermediate terminated, native state of enzyme regenerated

References

  1. Tsai SC et al. (2001), Proc Natl Acad Sci U S A, 98, 14808-14813. Crystal structure of the macrocycle-forming thioesterase domain of the erythromycin polyketide synthase: Versatility from a unique substrate channel. DOI:10.1073/pnas.011399198. PMID:11752428.
  2. Robbins T et al. (2016), Curr Opin Struct Biol, 41, 10-18. Structure and mechanism of assembly line polyketide synthases. DOI:10.1016/j.sbi.2016.05.009. PMID:27266330.
  3. Chen X et al. (2016), ACS Catal, 6, 4369-4378. Theoretical Studies on the Mechanism of Thioesterase-Catalyzed Macrocyclization in Erythromycin Biosynthesis. DOI:10.1021/acscatal.6b01154.

Step 1. His259 deprotonates Ser142 which activates it for nucleophilic attack on the C1 carbonyl carbon.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ser142A covalently attached
Ala143A (main-N) electrostatic stabiliser
Asp169A electrostatic stabiliser, modifies pKa
His259A proton acceptor
Ser142A proton donor, nucleophile

Chemical Components

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

Step 2. The oxyanion collapse resulting in the elimination of CoA which will accept a proton from the C13 OH

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ala143A (main-N) electrostatic stabiliser
Asp169A electrostatic stabiliser, modifies pKa
Ser142A covalently attached

Chemical Components

ingold: unimolecular elimination by the conjugate base, intermediate collapse, intermediate formation, overall product formed, proton transfer

Step 3. The deprotonated C11 oxygen then nucleophilically attacks the C1 carbonyl carbon.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ala143A (main-N) electrostatic stabiliser
Asp169A electrostatic stabiliser, modifies pKa
Ser142A covalently attached

Chemical Components

ingold: bimolecular nucleophilic addition, intermediate formation

Step 4. The oxyanion intiates an elimination which results in the cleavage of the acyl-enzyme bond and the released Ser142 will then accept a proton from His259.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ala143A (main-N) electrostatic stabiliser
Asp169A electrostatic stabiliser, modifies pKa
Ser142A proton acceptor
His259A proton donor
Ser142A nucleofuge

Chemical Components

ingold: unimolecular elimination by the conjugate base, enzyme-substrate complex cleavage, intermediate collapse, intermediate terminated, native state of enzyme regenerated, overall product formed, proton transfer
Download: Image, Marvin File

Step 1. His259 deprotonates Ser142 which activates it for nucleophilic attack on the C1 carbonyl carbon.

Step 2. The oxyanion collapse resulting in the elimination of CoA which will accept a proton from the C13 OH

Step 3. The deprotonated C11 oxygen then nucleophilically attacks the C1 carbonyl carbon.

Step 4. The oxyanion intiates an elimination which results in the cleavage of the acyl-enzyme bond and the released Ser142 will then accept a proton from His259.

Products.

Contributors

Mei Leung, Gemma L. Holliday, Charity Hornby