4CXK

G9 mutant of PAS, arylsulfatase from Pseudomonas Aeruginosa


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.86 Å
  • R-Value Free: 0.254 
  • R-Value Work: 0.208 
  • R-Value Observed: 0.210 

wwPDB Validation   3D Report Full Report


This is version 2.2 of the entry. See complete history


Literature

Evolutionary repurposing of a sulfatase: A new Michaelis complex leads to efficient transition state charge offset.

Miton, C.M.Jonas, S.Fischer, G.Duarte, F.Mohamed, M.F.van Loo, B.Kintses, B.Kamerlin, S.C.L.Tokuriki, N.Hyvonen, M.Hollfelder, F.

(2018) Proc Natl Acad Sci U S A 115: E7293-E7302

  • DOI: https://doi.org/10.1073/pnas.1607817115
  • Primary Citation of Related Structures:  
    4CXK, 4CXS, 4CXU, 4CYR, 4CYS, 5AJ9

  • PubMed Abstract: 

    The recruitment and evolutionary optimization of promiscuous enzymes is key to the rapid adaptation of organisms to changing environments. Our understanding of the precise mechanisms underlying enzyme repurposing is, however, limited: What are the active-site features that enable the molecular recognition of multiple substrates with contrasting catalytic requirements? To gain insights into the molecular determinants of adaptation in promiscuous enzymes, we performed the laboratory evolution of an arylsulfatase to improve its initially weak phenylphosphonate hydrolase activity. The evolutionary trajectory led to a 100,000-fold enhancement of phenylphosphonate hydrolysis, while the native sulfate and promiscuous phosphate mono- and diester hydrolyses were only marginally affected (≤50-fold). Structural, kinetic, and in silico characterizations of the evolutionary intermediates revealed that two key mutations, T50A and M72V, locally reshaped the active site, improving access to the catalytic machinery for the phosphonate. Measured transition state (TS) charge changes along the trajectory suggest the creation of a new Michaelis complex (E•S, enzyme-substrate), with enhanced leaving group stabilization in the TS for the promiscuous phosphonate ( β leaving group from -1.08 to -0.42). Rather than altering the catalytic machinery, evolutionary repurposing was achieved by fine-tuning the molecular recognition of the phosphonate in the Michaelis complex, and by extension, also in the TS. This molecular scenario constitutes a mechanistic alternative to adaptation solely based on enzyme flexibility and conformational selection. Instead, rapid functional transitions between distinct chemical reactions rely on the high reactivity of permissive active-site architectures that allow multiple substrate binding modes.


  • Organizational Affiliation

    Department of Biochemistry, University of Cambridge, CB2 1GA Cambridge, United Kingdom.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
ARYLSULFATASE
A, B
536Pseudomonas aeruginosaMutation(s): 8 
EC: 3.1.6.1
UniProt
Find proteins for P51691 (Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1))
Explore P51691 
Go to UniProtKB:  P51691
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP51691
Sequence Annotations
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.86 Å
  • R-Value Free: 0.254 
  • R-Value Work: 0.208 
  • R-Value Observed: 0.210 
  • Space Group: C 1 2 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 188.298α = 90
b = 67.061β = 94.04
c = 90.05γ = 90
Software Package:
Software NamePurpose
REFMACrefinement
autoPROCdata reduction
Aimlessdata scaling
PHASERphasing

Structure Validation

View Full Validation Report



Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2015-04-22
    Type: Initial release
  • Version 1.1: 2018-06-27
    Changes: Data collection, Derived calculations
  • Version 1.2: 2018-08-29
    Changes: Data collection, Database references
  • Version 2.0: 2019-04-24
    Changes: Data collection, Polymer sequence
  • Version 2.1: 2019-07-10
    Changes: Data collection
  • Version 2.2: 2023-12-20
    Changes: Data collection, Database references, Derived calculations, Other, Refinement description