3TC7

Crystal Structure of Engineered Protein. Northeast Structural Genomics Consortium Target OR62.


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.50 Å
  • R-Value Free: 0.193 
  • R-Value Work: 0.158 
  • R-Value Observed: 0.160 

wwPDB Validation   3D Report Full Report


This is version 1.3 of the entry. See complete history


Literature

Exploration of alternate catalytic mechanisms and optimization strategies for retroaldolase design.

Bjelic, S.Kipnis, Y.Wang, L.Pianowski, Z.Vorobiev, S.Su, M.Seetharaman, J.Xiao, R.Kornhaber, G.Hunt, J.F.Tong, L.Hilvert, D.Baker, D.

(2014) J Mol Biol 426: 256-271

  • DOI: https://doi.org/10.1016/j.jmb.2013.10.012
  • Primary Citation of Related Structures:  
    3TC6, 3TC7

  • PubMed Abstract: 

    Designed retroaldolases have utilized a nucleophilic lysine to promote carbon-carbon bond cleavage of β-hydroxy-ketones via a covalent Schiff base intermediate. Previous computational designs have incorporated a water molecule to facilitate formation and breakdown of the carbinolamine intermediate to give the Schiff base and to function as a general acid/base. Here we investigate an alternative active-site design in which the catalytic water molecule was replaced by the side chain of a glutamic acid. Five out of seven designs expressed solubly and exhibited catalytic efficiencies similar to previously designed retroaldolases for the conversion of 4-hydroxy-4-(6-methoxy-2-naphthyl)-2-butanone to 6-methoxy-2-naphthaldehyde and acetone. After one round of site-directed saturation mutagenesis, improved variants of the two best designs, RA114 and RA117, exhibited among the highest kcat (>10(-3)s(-1)) and kcat/KM (11-25M(-1)s(-1)) values observed for retroaldolase designs prior to comprehensive directed evolution. In both cases, the >10(5)-fold rate accelerations that were achieved are within 1-3 orders of magnitude of the rate enhancements reported for the best catalysts for related reactions, including catalytic antibodies (kcat/kuncat=10(6) to 10(8)) and an extensively evolved computational design (kcat/kuncat>10(7)). The catalytic sites, revealed by X-ray structures of optimized versions of the two active designs, are in close agreement with the design models except for the catalytic lysine in RA114. We further improved the variants by computational remodeling of the loops and yeast display selection for reactivity of the catalytic lysine with a diketone probe, obtaining an additional order of magnitude enhancement in activity with both approaches.


  • Organizational Affiliation

    Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Indole-3-glycerol phosphate synthase258Saccharolobus solfataricus P2Mutation(s): 16 
Gene Names: trpCSSO0895
EC: 4.1.1.48
UniProt
Find proteins for Q06121 (Saccharolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2))
Explore Q06121 
Go to UniProtKB:  Q06121
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupQ06121
Sequence Annotations
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.50 Å
  • R-Value Free: 0.193 
  • R-Value Work: 0.158 
  • R-Value Observed: 0.160 
  • Space Group: P 31 2 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 60.96α = 90
b = 60.96β = 90
c = 118.663γ = 120
Software Package:
Software NamePurpose
PHENIXrefinement
PDB_EXTRACTdata extraction
ADSCdata collection
HKL-2000data reduction
HKL-2000data scaling
BALBESphasing

Structure Validation

View Full Validation Report



Entry History 

Revision History  (Full details and data files)

  • Version 1.0: 2011-08-24
    Type: Initial release
  • Version 1.1: 2013-12-04
    Changes: Database references
  • Version 1.2: 2014-01-08
    Changes: Database references
  • Version 1.3: 2023-09-13
    Changes: Data collection, Database references, Derived calculations, Refinement description