5KJB

Synechocystis apocarotenoid oxygenase (ACO) mutant - Glu150Asp


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.81 Å
  • R-Value Free: 0.233 
  • R-Value Work: 0.202 
  • R-Value Observed: 0.203 

wwPDB Validation   3D Report Full Report


This is version 1.3 of the entry. See complete history


Literature

Key Residues for Catalytic Function and Metal Coordination in a Carotenoid Cleavage Dioxygenase.

Sui, X.Zhang, J.Golczak, M.Palczewski, K.Kiser, P.D.

(2016) J Biol Chem 291: 19401-19412

  • DOI: https://doi.org/10.1074/jbc.M116.744912
  • Primary Citation of Related Structures:  
    5KJA, 5KJB, 5KJD

  • PubMed Abstract: 

    Carotenoid cleavage dioxygenases (CCDs) are non-heme iron-containing enzymes found in all domains of life that generate biologically important apocarotenoids. Prior studies have revealed a critical role for a conserved 4-His motif in forming the CCD iron center. By contrast, the roles of other active site residues in catalytic function, including maintenance of the stringent regio- and stereo-selective cleavage activity, typically exhibited by these enzymes have not been thoroughly investigated. Here, we examined the functional and structural importance of active site residues in an apocarotenoid-cleaving oxygenase (ACO) from Synechocystis Most active site substitutions variably lowered maximal catalytic activity without markedly affecting the Km value for the all-trans-8'-apocarotenol substrate. Native C15-C15' cleavage activity was retained in all ACO variants examined suggesting that multiple active site residues contribute to the enzyme's regioselectivity. Crystallographic analysis of a nearly inactive W149A-substituted ACO revealed marked disruption of the active site structure, including loss of iron coordination by His-238 apparently from an altered conformation of the conserved second sphere Glu-150 residue. Gln- and Asp-150-substituted versions of ACO further confirmed the structural/functional requirement for a Glu side chain at this position, which is homologous to Glu-148 in RPE65, a site in which substitution to Asp has been associated with loss of enzymatic function in Leber congenital amaurosis. The novel links shown here between ACO active site structure and catalytic activity could be broadly applicable to other CCD members and provide insights into the molecular pathogenesis of vision loss associated with an RPE65 point mutation.


  • Organizational Affiliation

    From the Department of Pharmacology, School of Medicine, Case Western Reserve University and.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Apocarotenoid-15,15'-oxygenase
A, B, C, D, E
490Synechocystis sp. PCC 6803 substr. KazusaMutation(s): 1 
Gene Names: sll1541
EC: 1.13.11.75
UniProt
Find proteins for P74334 (Synechocystis sp. (strain PCC 6803 / Kazusa))
Explore P74334 
Go to UniProtKB:  P74334
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP74334
Sequence Annotations
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.81 Å
  • R-Value Free: 0.233 
  • R-Value Work: 0.202 
  • R-Value Observed: 0.203 
  • Space Group: P 21 21 21
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 118.42α = 90
b = 124.95β = 90
c = 203.97γ = 90
Software Package:
Software NamePurpose
REFMACrefinement
XDSdata reduction
XSCALEdata scaling
PHASERphasing

Structure Validation

View Full Validation Report



Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2016-08-03
    Type: Initial release
  • Version 1.1: 2016-08-10
    Changes: Database references
  • Version 1.2: 2016-09-21
    Changes: Database references
  • Version 1.3: 2023-09-27
    Changes: Data collection, Database references, Derived calculations, Refinement description