Naringenin-chalcone synthase

 

Chalcone synthase (CHS) is pivotal for the biosynthesis of flavonoid antimicrobial phytoalexins and anthocyanin pigments in plants. It produces chalcone by condensing one p-coumaroyl- and three malonyl-coenzyme A thioesters into a polyketide reaction intermediate that cyclises without the involvement of specific catalytic residues.

 

Reference Protein and Structure

Sequence
P30074 UniProt (2.3.1.74) IPR011141 (Sequence Homologues) (PDB Homologues)
Biological species
Medicago sativa (Alfalfa) Uniprot
PDB
1cgk - CHALCONE SYNTHASE FROM ALFALFA COMPLEXED WITH NARINGENIN (1.84 Å) PDBe PDBsum 1cgk
Catalytic CATH Domains
3.40.47.10 CATHdb (see all for 1cgk)
Click To Show Structure

Enzyme Reaction (EC:2.3.1.74)

hydron
CHEBI:15378ChEBI
+
4-coumaroyl-CoA(4-)
CHEBI:57355ChEBI
+
malonyl-CoA(5-)
CHEBI:57384ChEBI
→
2',4,4',6'-tetrahydroxychalcone(1-)
CHEBI:77645ChEBI
+
coenzyme A(4-)
CHEBI:57287ChEBI
+
carbon dioxide
CHEBI:16526ChEBI
Alternative enzyme names: 6'-deoxychalcone synthase, CHS, DOCS, Chalcone synthase, Chalcone synthetase, Flavanone synthase, Flavonone synthase, Naringenin-chalcone synthase,

Enzyme Mechanism

Introduction

Binding of p-coumaroyl-CoA initiates the reaction. The thiolate of Cys164 attacks the thioester carbonyl, resulting in transfer of the coumaroyl moiety to the cysteine side chain. Asn336 hydrogen bonds with the thioester carbonyl, further stabilizing formation of the tetrahedral reaction intermediate. CoA then dissociates from the enzyme, leaving a coumaroyl-thioester at Cys164. Malonyl-CoA binds and positions the bridging carbon of the malonyl moiety near the carbonyl of the enzyme-bound coumaroyl thioester. Asn336 orients the thioester carbonyl of malonyl-CoA near His303 with Phe215, providing a nonpolar environment for the terminal carboxylate that facilitates decarboxylation. His303 and Asn336 interact with the substrate's thioester carbonyl, creating an efficient oxyanion hole that stabilizes the developing negative charge during the decarboxylation step through stabilization of the enol tautomer of the acetyl anion. Attack of the carbanion on the carbonyl of the enzyme-bound coumaroyl thioester releases the thiolate anion of Cys164 and transfers the coumaroyl group to the acetyl moiety of the CoA thioester. Hydrogen bonds from His303 and Asn336 would stabilize the tetrahedral transition state of this reaction. Recapture of the elongated coumaroyl-acetyl-diketide-CoA by Cys164 and release of CoA set the stage for two additional rounds of elongation, resulting in formation of the final tetraketide reaction intermediate. The final step in chalcone formation involves an intramolecular Claisen condensation encompassing the three acetate units derived from three malonyl-CoAs. During cyclization, the nucleophilic methylene group nearest the coumaroyl moiety attacks the carbonyl carbon of the thioester linked to Cys164. Ring closure is proposed to proceed through an internal proton transfer from the nucleophilic carbon to the carbonyl oxygen. Breakdown of this tetrahedral intermediate expels the newly cyclized ring system from Cys164. Subsequent aromatization of the trione ring through a second series of facile internal proton transfers yields chalcone.

Catalytic Residues Roles

UniProt PDB* (1cgk)
Phe215 Phe215A Provides a non-polar environment which encourages the formation of CO2 during decarboxylation. electrostatic destabiliser, polar/non-polar interaction
His303 His303A The protonated residue forms a stable thiolate-imidazolium ion pair thus activating Cys164. This group also forms part of an oxyanion hole during decarboxylation. hydrogen bond donor, electrostatic stabiliser
Cys164 Cys164A Serves as the nucleophile for polyketide formation, forming a enzyme adduct and releasing CoA. activator, covalently attached, nucleofuge, nucleophile
Asn336 Asn336A Forms part of an oxyanion hole during decarboxylation. hydrogen bond donor, 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

bimolecular nucleophilic addition, overall reactant used, intermediate formation, enzyme-substrate complex formation, elimination (not covered by the Ingold mechanisms), intermediate collapse, overall product formed, intramolecular elimination, decarboxylation, enzyme-substrate complex cleavage, intramolecular nucleophilic addition, proton transfer, cyclisation, inferred reaction step, native state of enzyme regenerated, intramolecular rearrangement

References

  1. Jez JM et al. (2000), Biochemistry, 39, 890-902. Dissection of Malonyl-Coenzyme A Decarboxylation from Polyketide Formation in the Reaction Mechanism of a Plant Polyketide Synthase†. DOI:10.1021/bi991489f. PMID:10653632.
  2. Healy EF et al. (2018), Biochem Biophys Res Commun, 497, 1123-1128. A unified mechanism for plant polyketide biosynthesis derived from in silico modeling. DOI:10.1016/j.bbrc.2018.02.190. PMID:29496450.
  3. Li D et al. (2009), Theor Chem Acc, 122, 157-166. A quantum mechanics study on the reaction mechanism of chalcone formation from p-coumaroyl-CoA and malonyl-CoA catalyzed by chalcone synthase. DOI:10.1007/s00214-008-0495-7.
  4. Abe I et al. (2000), J Am Chem Soc, 122, 11242-11243. Substrate Specificity of Chalcone Synthase:  Enzymatic Formation of Unnatural Polyketides from Synthetic Cinnamoyl-CoA Analogues. DOI:10.1021/ja0027113.
  5. Ferrer JL et al. (1999), Nat Struct Biol, 6, 775-784. Structure of chalcone synthase and the molecular basis of plant polyketide biosynthesis. DOI:10.1038/11553. PMID:10426957.
  6. Tropf S et al. (1995), J Biol Chem, 270, 7922-7928. Reaction mechanisms of homodimeric plant polyketide synthase (stilbenes and chalcone synthase). A single active site for the condensing reaction is sufficient for synthesis of stilbenes, chalcones, and 6'-deoxychalcones. PMID:7713888.

Step 1. The anionic Cys164 attacks at the 4-coumaroyl-CoA thio-carbonyl, forming an anionic tetrahedral intermediate.

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Catalytic Residues Roles

Residue Roles
Asn336A electrostatic stabiliser, hydrogen bond donor
His303A electrostatic stabiliser, hydrogen bond donor
Cys164A activator, nucleophile

Chemical Components

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

Step 2. The tetrahedral intermediate collapses, eliminating coenzyme A and foming a covalently bonded substrate-enzyme intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn336A electrostatic stabiliser, hydrogen bond donor
His303A electrostatic stabiliser, hydrogen bond donor
Cys164A activator, covalently attached
Phe215A polar/non-polar interaction, electrostatic destabiliser

Chemical Components

elimination (not covered by the Ingold mechanisms), intermediate collapse, overall product formed

Step 3. The polar-nonpolar interaction of Phe215 and malonyl-CoA drives decarboxylation to form the enolate intermediate which is stabilised through hydrogen bonding with Asn336.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn336A electrostatic stabiliser, hydrogen bond donor
His303A hydrogen bond donor
Cys164A activator, covalently attached
Phe215A polar/non-polar interaction, electrostatic destabiliser

Chemical Components

ingold: intramolecular elimination, intermediate formation, overall reactant used, decarboxylation, overall product formed

Step 4. The enolate intermediate attacks the thio-carbonyl, forming an anionic tetrahedral intermediate while extending the carbon chain by two units.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn336A electrostatic stabiliser, hydrogen bond donor
His303A hydrogen bond donor
Cys164A activator, covalently attached

Chemical Components

ingold: bimolecular nucleophilic addition, intermediate formation

Step 5. The tetrahedral intermediate collapses, eliminating the Cys164 sidechain. The position of Phe215 postulates its involvement in driving the decomposition of the tetrahedral intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn336A electrostatic stabiliser, hydrogen bond donor
His303A hydrogen bond donor
Cys164A activator
Phe215A polar/non-polar interaction
Cys164A nucleofuge

Chemical Components

elimination (not covered by the Ingold mechanisms), intermediate formation, enzyme-substrate complex cleavage

Step 6. Cys164 attacks at the thio-carbonyl, reforming an enzyme-substrate tetrahedral intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn336A electrostatic stabiliser, hydrogen bond donor
His303A hydrogen bond donor
Cys164A activator, nucleophile

Chemical Components

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

Step 7. The tetrahedral intermediate collapses, eliminating coenzyme A and forming the enzyme-bonded mono-ketide product.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn336A electrostatic stabiliser, hydrogen bond donor
His303A hydrogen bond donor
Cys164A activator, covalently attached

Chemical Components

elimination (not covered by the Ingold mechanisms), intermediate formation, overall product formed

Step 8. The polar-nonpolar interaction of Phe215 and malonyl-CoA drives decarboxylation to form the enolate intermediate which is stabilised through hydrogen bonding with Asn336.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn336A electrostatic stabiliser, hydrogen bond donor
His303A hydrogen bond donor
Cys164A activator, covalently attached
Phe215A polar/non-polar interaction, electrostatic destabiliser

Chemical Components

ingold: intramolecular elimination, intermediate formation, overall reactant used, decarboxylation, overall product formed

Step 9. The enolate intermediate attacks the thio-carbonyl, forming an anionic tetrahedral intermediate while extending the carbon chain by two units.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn336A electrostatic stabiliser, hydrogen bond donor
His303A hydrogen bond donor
Cys164A activator, covalently attached

Chemical Components

ingold: bimolecular nucleophilic addition, intermediate formation

Step 10. The tetrahedral intermediate collapses, eliminating the Cys164 sidechain. The position of Phe215 postulates its involvement in driving the decomposition of the tetrahedral intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn336A electrostatic stabiliser, hydrogen bond donor
His303A hydrogen bond donor
Cys164A activator
Phe215A polar/non-polar interaction
Cys164A nucleofuge

Chemical Components

elimination (not covered by the Ingold mechanisms), intermediate formation, enzyme-substrate complex cleavage

Step 11. Cys164 attacks at the thio-carbonyl, reforming an enzyme-substrate tetrahedral intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn336A electrostatic stabiliser, hydrogen bond donor
His303A hydrogen bond donor
Cys164A activator, nucleophile

Chemical Components

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

Step 12. The tetrahedral intermediate collapses, eliminating coenzyme A and forming the enzyme-bonded mono-ketide product.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn336A electrostatic stabiliser, hydrogen bond donor
His303A hydrogen bond donor
Cys164A activator, covalently attached

Chemical Components

elimination (not covered by the Ingold mechanisms), intermediate formation, overall product formed

Step 13. The polar-nonpolar interaction of Phe215 and malonyl-CoA drives decarboxylation to form the enolate intermediate which is stabilised through hydrogen bonding with Asn336.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Phe215A electrostatic destabiliser, polar/non-polar interaction
Cys164A covalently attached, activator
His303A hydrogen bond donor
Asn336A hydrogen bond donor, electrostatic stabiliser

Chemical Components

overall product formed, decarboxylation, overall reactant used, intermediate formation, ingold: intramolecular elimination

Step 14. The enolate intermediate attacks the thio-carbonyl, forming an anionic tetrahedral intermediate while extending the carbon chain by two units.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys164A covalently attached, activator
His303A hydrogen bond donor
Asn336A hydrogen bond donor, electrostatic stabiliser

Chemical Components

intermediate formation, ingold: bimolecular nucleophilic addition

Step 15. The tetrahedral intermediate collapses, eliminating the Cys164 sidechain. The position of Phe215 postulates its involvement in driving the decomposition of the tetrahedral intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Phe215A polar/non-polar interaction
Cys164A activator
His303A hydrogen bond donor
Asn336A hydrogen bond donor, electrostatic stabiliser
Cys164A nucleofuge

Chemical Components

enzyme-substrate complex cleavage, intermediate formation, elimination (not covered by the Ingold mechanisms)

Step 16. Cys164 attacks at the thio-carbonyl, reforming an enzyme-substrate tetrahedral intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys164A activator
His303A hydrogen bond donor
Asn336A hydrogen bond donor, electrostatic stabiliser
Cys164A nucleophile

Chemical Components

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

Step 17. The tetrahedral intermediate collapses, eliminating coenzyme A and forming the enzyme-bonded mono-ketide product.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys164A covalently attached, activator
His303A hydrogen bond donor
Asn336A hydrogen bond donor, electrostatic stabiliser

Chemical Components

overall product formed, intermediate formation, elimination (not covered by the Ingold mechanisms)

Step 18. An intramolecular proton-rearrangment occurs during cyclisation.

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Catalytic Residues Roles

Residue Roles
Asn336A electrostatic stabiliser, hydrogen bond donor
His303A hydrogen bond donor
Cys164A activator, covalently attached

Chemical Components

ingold: intramolecular nucleophilic addition, proton transfer, intermediate formation, cyclisation

Step 19. The Cys164 side chain is eliminated, resulting in the formation of a cyclic tri-ketide. In the absence of chalcone isomerase, the enzyme-bound polyketide undergoes non-stereospecific C-ring closure [DOI:10.1021/ja0027113]. The involvement of water in abstracting a proton, as shown here, is inferred.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn336A electrostatic stabiliser, hydrogen bond donor
His303A hydrogen bond donor
Cys164A activator, covalently attached, nucleofuge

Chemical Components

ingold: intramolecular nucleophilic addition, intermediate formation, inferred reaction step

Step 20. The triketide ring undergoes intramolecular proton transfer to give an aromatic ring.

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Catalytic Residues Roles

Residue Roles
Asn336A hydrogen bond donor
His303A hydrogen bond donor
Cys164A activator

Chemical Components

proton transfer, native state of enzyme regenerated, overall product formed, intramolecular rearrangement
Download: Image, Marvin File

Step 1. The anionic Cys164 attacks at the 4-coumaroyl-CoA thio-carbonyl, forming an anionic tetrahedral intermediate.

Step 2. The tetrahedral intermediate collapses, eliminating coenzyme A and foming a covalently bonded substrate-enzyme intermediate.

Step 3. The polar-nonpolar interaction of Phe215 and malonyl-CoA drives decarboxylation to form the enolate intermediate which is stabilised through hydrogen bonding with Asn336.

Step 4. The enolate intermediate attacks the thio-carbonyl, forming an anionic tetrahedral intermediate while extending the carbon chain by two units.

Step 5. The tetrahedral intermediate collapses, eliminating the Cys164 sidechain. The position of Phe215 postulates its involvement in driving the decomposition of the tetrahedral intermediate.

Step 6. Cys164 attacks at the thio-carbonyl, reforming an enzyme-substrate tetrahedral intermediate.

Step 7. The tetrahedral intermediate collapses, eliminating coenzyme A and forming the enzyme-bonded mono-ketide product.

Step 8. The polar-nonpolar interaction of Phe215 and malonyl-CoA drives decarboxylation to form the enolate intermediate which is stabilised through hydrogen bonding with Asn336.

Step 9. The enolate intermediate attacks the thio-carbonyl, forming an anionic tetrahedral intermediate while extending the carbon chain by two units.

Step 10. The tetrahedral intermediate collapses, eliminating the Cys164 sidechain. The position of Phe215 postulates its involvement in driving the decomposition of the tetrahedral intermediate.

Step 11. Cys164 attacks at the thio-carbonyl, reforming an enzyme-substrate tetrahedral intermediate.

Step 12. The tetrahedral intermediate collapses, eliminating coenzyme A and forming the enzyme-bonded mono-ketide product.

Step 13. The polar-nonpolar interaction of Phe215 and malonyl-CoA drives decarboxylation to form the enolate intermediate which is stabilised through hydrogen bonding with Asn336.

Step 14. The enolate intermediate attacks the thio-carbonyl, forming an anionic tetrahedral intermediate while extending the carbon chain by two units.

Step 15. The tetrahedral intermediate collapses, eliminating the Cys164 sidechain. The position of Phe215 postulates its involvement in driving the decomposition of the tetrahedral intermediate.

Step 16. Cys164 attacks at the thio-carbonyl, reforming an enzyme-substrate tetrahedral intermediate.

Step 17. The tetrahedral intermediate collapses, eliminating coenzyme A and forming the enzyme-bonded mono-ketide product.

Step 18. An intramolecular proton-rearrangment occurs during cyclisation.

Step 19. The Cys164 side chain is eliminated, resulting in the formation of a cyclic tri-ketide. In the absence of chalcone isomerase, the enzyme-bound polyketide undergoes non-stereospecific C-ring closure [DOI:10.1021/ja0027113]. The involvement of water in abstracting a proton, as shown here, is inferred.

Step 20. The triketide ring undergoes intramolecular proton transfer to give an aromatic ring.

Products.

Contributors

Sophie T. Williams, Nozomi Nagano, Craig Porter, Gemma L. Holliday, Charity Hornby