Chalcone isomerase

 

Chalcone isomerase is required in plant flavonoid biosynthesis, catalysing the cyclisation of chalcone with a 1:100,000 preference for the S-isomer over the R- isomer. This pathway is well characterised, and so the flavonoid biosynthetic enzymes are attractive targets for metabolic engineering.

 

Reference Protein and Structure

Sequence
P28012 UniProt (5.5.1.6) IPR016087 (Sequence Homologues) (PDB Homologues)
Biological species
Medicago sativa (Alfalfa) Uniprot
PDB
1eyq - Chalcone isomerase and naringenin (1.85 Å) PDBe PDBsum 1eyq
Catalytic CATH Domains
3.50.70.10 CATHdb (see all for 1eyq)
Cofactors
Water (2)
Click To Show Structure

Enzyme Reaction (EC:5.5.1.6)

2'-hydroxychalcone
CHEBI:27916ChEBI
→
flavanone
CHEBI:5070ChEBI
Alternative enzyme names: Chalcone--flavanone isomerase, Flavanone lyase (decyclizing),

Enzyme Mechanism

Introduction

The overall mechanism is a Michael addition; the intra-molecular nucleophilic attack of a hydroxyl at a carbonyl in the chalcone substrate, through a six member transition state to form (2S)-naringenin. Modelling studies implicate an electron flow towards the C alpha atom, forming a corresponding carbanion transition state. The charge on this carbon atom increases substantially more than the charge found on the carbonyl oxygen atom. The proximity of the C alpha to a positively charged lysine, and the resulting stabilisation is thought to be the largest contribution to the enzyme's catalytic power. This Lys is thought to then act as a general acid to the anionic transition state through a water molecule. The ability of the active site to accommodate and stabilise the charge distribution of the transition state drives catalysis.

The pH dependence of the non-enzymatic and Chalcone Isomerase (CHI) catalysed reactions implies that a significant portion of the physiologic substrate pool is found in the reactive deprotonated form [PMID:11698411]. Previous studies showed that the cyclisation reaction catalysed by wild type CHI is approximately 90% diffusion controlled [PMID:11955065,PMID:11698411]. Asn113 and Thr190 orient the substrate at the active site and position the reactive 2'-oxyanion of the substrate in proximity to the alpha beta-unsaturated double bond for the intrmolecular cyclisation reaction [PMID:11955065].

Catalytic Residues Roles

UniProt PDB* (1eyq)
Arg36 Arg36A Interacts with the 4’-hydroxyl group of isoliquiritigenin (a similar substrate which the enzyme also converts to a flavanone by the same mechanism)
Lys97 Lys97A The positively charged residue polarises a close proximity water molecule, which then acts to stabilise the carbanionic transition state. The electrostatic potential is important in lowering the energy of the transition state relative to the reactant. The residue is also necessary to induce protonation of the anion by the water molecule through hydrogen bond interactions. modifies pKa
Tyr106 Tyr106A The residue establishes a hydrogen bond with the 6' hydroxyl group of the substrate through a water molecule. This interaction is significantly stronger in the transition state than in the reactant state, making a favourable interaction to lower the free energy barrier. activator, hydrogen bond donor
Thr48 Thr48A The residue hydroxyl interacts with the O7 of the substrate (the carbonyl oxygen), and orientates the transition state towards the Lys 97 residue. Mutation of Thr 48 to Ala results in a loss of catalytic function by roughly 1000 fold. 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

intramolecular nucleophilic addition, proton transfer, overall reactant used, overall product formed

References

  1. Jez JM et al. (2002), J Biol Chem, 277, 1361-1369. Reaction Mechanism of Chalcone Isomerase: pH DEPENDENCE, DIFFUSION CONTROL, AND PRODUCT BINDING DIFFERENCES. DOI:10.1074/jbc.m109224200. PMID:11698411.
  2. Park SH et al. (2018), PLoS One, 13, e0192415-. Crystal structure and enzymatic properties of chalcone isomerase from the Antarctic vascular plant Deschampsia antarctica Desv. DOI:10.1371/journal.pone.0192415. PMID:29394293.
  3. Ruiz-Pernía JJ et al. (2007), J Am Chem Soc, 129, 9117-9124. Enzymatic effects on reactant and transition states. The case of chalcone isomerase. DOI:10.1021/ja071720+. PMID:17602559.
  4. Hur S et al. (2004), Proc Natl Acad Sci U S A, 101, 2730-2735. Transition state stabilization by general acid catalysis, water expulsion, and enzyme reorganization in Medicago savita chalcone isomerase. DOI:10.1073/pnas.0308264100. PMID:14978275.
  5. Jez JM et al. (2002), Biochemistry, 41, 5168-5176. Role of Hydrogen Bonds in the Reaction Mechanism of Chalcone Isomerase†. DOI:10.1021/bi0255266. PMID:11955065.
  6. Jez JM et al. (2000), Nat Struct Biol, 7, 786-791. Structure and mechanism of the evolutionarily unique plant enzyme chalcone isomerase. DOI:10.1038/79025. PMID:10966651.

Step 1. The chalcone substrate undergoes an intramolecular nucleophilic addition, and deprotonates a water molecule to generate the flavanone product. The cyclisation reaction proceeds through a transition state in which charge delocalisation results in the transient protonation of the transition state enolate by the water molecule hydrogen bonded to Tyr106 [PMID:11955065,PMID:11698411].

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Catalytic Residues Roles

Residue Roles
Tyr106A activator, hydrogen bond donor
Thr48A hydrogen bond donor, electrostatic stabiliser
Lys97A modifies pKa

Chemical Components

ingold: intramolecular nucleophilic addition, proton transfer, overall reactant used, overall product formed
Download: Image, Marvin File

Step 1. The chalcone substrate undergoes an intramolecular nucleophilic addition, and deprotonates a water molecule to generate the flavanone product. The cyclisation reaction proceeds through a transition state in which charge delocalisation results in the transient protonation of the transition state enolate by the water molecule hydrogen bonded to Tyr106 [PMID:11955065,PMID:11698411].

Products.

Contributors

Gemma L. Holliday, Daniel E. Almonacid, James W. Murray, Craig Porter, James Willey