3WY0

The I375W mutant of CsyB complexed with CoA-SH


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.00 Å
  • R-Value Free: 0.204 
  • R-Value Work: 0.171 
  • R-Value Observed: 0.173 

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Ligand Structure Quality Assessment 


This is version 1.2 of the entry. See complete history


Literature

Structural basis for the formation of acylalkylpyrones from two beta-ketoacyl units by the fungal type III polyketide synthase CsyB.

Mori, T.Yang, D.Matsui, T.Hashimoto, M.Morita, H.Fujii, I.Abe, I.

(2015) J Biol Chem 290: 5214-5225

  • DOI: https://doi.org/10.1074/jbc.M114.626416
  • Primary Citation of Related Structures:  
    3WXY, 3WXZ, 3WY0

  • PubMed Abstract: 

    The acylalkylpyrone synthase CsyB from Aspergillus oryzae catalyzes the one-pot formation of the 3-acyl-4-hydroxy-6-alkyl-α-pyrone scaffold from acetoacetyl-CoA, fatty acyl-CoA, and malonyl-CoA. This is the first type III polyketide synthase that performs not only the polyketide chain elongation but also the condensation of two β-ketoacyl units. The crystal structures of wild-type CsyB and its I375F and I375W mutants were solved at 1.7-, 2.3-, and 2.0-Å resolutions, respectively. The crystal structures revealed a unique active site architecture featuring a hitherto unidentified novel pocket for accommodation of the acetoacetyl-CoA starter in addition to the conventional elongation/cyclization pocket with the Cys-His-Asn catalytic triad and the long hydrophobic tunnel for binding the fatty acyl chain. The structures also indicated the presence of a putative nucleophilic water molecule activated by the hydrogen bond networks with His-377 and Cys-155 at the active site center. Furthermore, an in vitro enzyme reaction confirmed that the (18)O atom of the H2(18)O molecule is enzymatically incorporated into the final product. These observations suggested that the enzyme reaction is initiated by the loading of acetoacetyl-CoA onto Cys-155, and subsequent thioester bond cleavage by the nucleophilic water generates the β-keto acid intermediate, which is placed within the novel pocket. The second β-ketoacyl unit is then produced by polyketide chain elongation of fatty acyl-CoA with one molecule of malonyl-CoA, and the condensation with the β-keto acid generates the final products. Indeed, steric modulation of the novel pocket by the structure-based I375F and I375W mutations resulted in altered specificities for the chain lengths of the substrates.


  • Organizational Affiliation

    From the Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.


Macromolecules
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Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Putative uncharacterized protein csyB
A, B
417Aspergillus oryzaeMutation(s): 1 
Gene Names: csyB
UniProt
Find proteins for Q2U852 (Aspergillus oryzae (strain ATCC 42149 / RIB 40))
Explore Q2U852 
Go to UniProtKB:  Q2U852
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupQ2U852
Sequence Annotations
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  • Reference Sequence
Small Molecules
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.00 Å
  • R-Value Free: 0.204 
  • R-Value Work: 0.171 
  • R-Value Observed: 0.173 
  • Space Group: P 1 21 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 70.13α = 90
b = 104.56β = 114.33
c = 73.66γ = 90
Software Package:
Software NamePurpose
XDSdata scaling
PHASERphasing
PHENIXrefinement
XDSdata reduction
XSCALEdata scaling

Structure Validation

View Full Validation Report



Ligand Structure Quality Assessment 


Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2015-01-14
    Type: Initial release
  • Version 1.1: 2019-12-25
    Changes: Database references
  • Version 1.2: 2023-11-08
    Changes: Data collection, Database references, Derived calculations, Refinement description