3UAK

Crystal Structure of De Novo designed cysteine esterase ECH14, Northeast Structural Genomics Consortium Target OR54


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 3.23 Å
  • R-Value Free: 0.287 
  • R-Value Work: 0.210 
  • R-Value Observed: 0.218 

wwPDB Validation   3D Report Full Report


This is version 1.4 of the entry. See complete history


Literature

Computational design of catalytic dyads and oxyanion holes for ester hydrolysis.

Richter, F.Blomberg, R.Khare, S.D.Kiss, G.Kuzin, A.P.Smith, A.J.Gallaher, J.Pianowski, Z.Helgeson, R.C.Grjasnow, A.Xiao, R.Seetharaman, J.Su, M.Vorobiev, S.Lew, S.Forouhar, F.Kornhaber, G.J.Hunt, J.F.Montelione, G.T.Tong, L.Houk, K.N.Hilvert, D.Baker, D.

(2012) J Am Chem Soc 134: 16197-16206

  • DOI: https://doi.org/10.1021/ja3037367
  • Primary Citation of Related Structures:  
    3U13, 3U1O, 3U1V, 3UAK

  • PubMed Abstract: 

    Nucleophilic catalysis is a general strategy for accelerating ester and amide hydrolysis. In natural active sites, nucleophilic elements such as catalytic dyads and triads are usually paired with oxyanion holes for substrate activation, but it is difficult to parse out the independent contributions of these elements or to understand how they emerged in the course of evolution. Here we explore the minimal requirements for esterase activity by computationally designing artificial catalysts using catalytic dyads and oxyanion holes. We found much higher success rates using designed oxyanion holes formed by backbone NH groups rather than by side chains or bridging water molecules and obtained four active designs in different scaffolds by combining this motif with a Cys-His dyad. Following active site optimization, the most active of the variants exhibited a catalytic efficiency (k(cat)/K(M)) of 400 M(-1) s(-1) for the cleavage of a p-nitrophenyl ester. Kinetic experiments indicate that the active site cysteines are rapidly acylated as programmed by design, but the subsequent slow hydrolysis of the acyl-enzyme intermediate limits overall catalytic efficiency. Moreover, the Cys-His dyads are not properly formed in crystal structures of the designed enzymes. These results highlight the challenges that computational design must overcome to achieve high levels of activity.


  • Organizational Affiliation

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


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
De Novo designed cysteine esterase ECH14
A, B
406synthetic constructMutation(s): 0 
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
Sequence Annotations
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  • Reference Sequence
Small Molecules
Modified Residues  1 Unique
IDChains TypeFormula2D DiagramParent
MSE
Query on MSE
A, B
L-PEPTIDE LINKINGC5 H11 N O2 SeMET
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 3.23 Å
  • R-Value Free: 0.287 
  • R-Value Work: 0.210 
  • R-Value Observed: 0.218 
  • Space Group: P 21 21 21
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 67.645α = 90
b = 81.814β = 90
c = 159.714γ = 90
Software Package:
Software NamePurpose
PHENIXrefinement
PDB_EXTRACTdata extraction
ADSCdata collection
DENZOdata reduction
SCALEPACKdata scaling
BALBESphasing
REFMACrefinement

Structure Validation

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Entry History 

Revision History  (Full details and data files)

  • Version 1.0: 2011-12-07
    Type: Initial release
  • Version 1.1: 2013-12-11
    Changes: Database references
  • Version 1.2: 2019-07-17
    Changes: Data collection, Derived calculations, Refinement description
  • Version 1.3: 2023-09-13
    Changes: Data collection, Database references, Refinement description
  • Version 1.4: 2023-12-06
    Changes: Data collection