1D6H

CHALONE SYNTHASE (N336A MUTANT COMPLEXED WITH COA)


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.15 Å
  • R-Value Free: 0.257 
  • R-Value Work: 0.184 

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Ligand Structure Quality Assessment 


This is version 1.4 of the entry. See complete history


Literature

Dissection of malonyl-coenzyme A decarboxylation from polyketide formation in the reaction mechanism of a plant polyketide synthase.

Jez, J.M.Ferrer, J.L.Bowman, M.E.Dixon, R.A.Noel, J.P.

(2000) Biochemistry 39: 890-902

  • DOI: https://doi.org/10.1021/bi991489f
  • Primary Citation of Related Structures:  
    1D6F, 1D6H, 1D6I

  • PubMed Abstract: 

    Chalcone synthase (CHS) catalyzes formation of the phenylpropanoid chalcone from one p-coumaroyl-CoA and three malonyl-coenzyme A (CoA) thioesters. The three-dimensional structure of CHS [Ferrer, J.-L., Jez, J. M., Bowman, M. E., Dixon, R. A., and Noel, J. P. (1999) Nat. Struct. Biol. 6, 775-784] suggests that four residues (Cys164, Phe215, His303, and Asn336) participate in the multiple decarboxylation and condensation reactions catalyzed by this enzyme. Here, we functionally characterize 16 point mutants of these residues for chalcone production, malonyl-CoA decarboxylation, and the ability to bind CoA and acetyl-CoA. Our results confirm Cys164's role as the active-site nucleophile in polyketide formation and elucidate the importance of His303 and Asn336 in the malonyl-CoA decarboxylation reaction. We suggest that Phe215 may help orient substrates at the active site during elongation of the polyketide intermediate. To better understand the structure-function relationships in some of these mutants, we also determined the crystal structures of the CHS C164A, H303Q, and N336A mutants refined to 1.69, 2.0, and 2.15 A resolution, respectively. The structure of the C164A mutant reveals that the proposed oxyanion hole formed by His303 and Asn336 remains undisturbed, allowing this mutant to catalyze malonyl-CoA decarboxylation without chalcone formation. The structures of the H303Q and N336A mutants support the importance of His303 and Asn336 in polarizing the thioester carbonyl of malonyl-CoA during the decarboxylation reaction. In addition, both of these residues may also participate in stabilizing the tetrahedral transition state during polyketide elongation. Conservation of the catalytic functions of the active-site residues may occur across a wide variety of condensing enzymes, including other polyketide and fatty acid synthases.


  • Organizational Affiliation

    Structural Biology Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, California 92037, USA.


Macromolecules
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Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
CHALCONE SYNTHASE387Medicago sativaMutation(s): 1 
EC: 2.3.1.74
UniProt
Find proteins for P30074 (Medicago sativa)
Explore P30074 
Go to UniProtKB:  P30074
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP30074
Sequence Annotations
Expand
  • Reference Sequence
Small Molecules
Modified Residues  1 Unique
IDChains TypeFormula2D DiagramParent
CSD
Query on CSD
A
L-PEPTIDE LINKINGC3 H7 N O4 SCYS
Binding Affinity Annotations 
IDSourceBinding Affinity
COA Binding MOAD:  1D6H Kd: 128 (nM) from 1 assay(s)
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.15 Å
  • R-Value Free: 0.257 
  • R-Value Work: 0.184 
  • Space Group: P 32 2 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 97.361α = 90
b = 97.361β = 90
c = 65.207γ = 120
Software Package:
Software NamePurpose
CCP4model building
REFMACrefinement
DENZOdata reduction
SCALEPACKdata scaling
CCP4phasing

Structure Validation

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Ligand Structure Quality Assessment 


Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2000-02-03
    Type: Initial release
  • Version 1.1: 2008-04-27
    Changes: Version format compliance
  • Version 1.2: 2011-07-13
    Changes: Derived calculations, Version format compliance
  • Version 1.3: 2018-01-31
    Changes: Experimental preparation
  • Version 1.4: 2021-11-03
    Changes: Database references, Derived calculations