5TPQ

E. coli alkaline phosphatase D101A, D153A, R166S, E322A, K328A mutant


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.45 Å
  • R-Value Free: 0.219 
  • R-Value Work: 0.165 
  • R-Value Observed: 0.167 

wwPDB Validation   3D Report Full Report


This is version 1.5 of the entry. See complete history


Literature

Differential catalytic promiscuity of the alkaline phosphatase superfamily bimetallo core reveals mechanistic features underlying enzyme evolution.

Sunden, F.AlSadhan, I.Lyubimov, A.Doukov, T.Swan, J.Herschlag, D.

(2017) J Biol Chem 292: 20960-20974

  • DOI: https://doi.org/10.1074/jbc.M117.788240
  • Primary Citation of Related Structures:  
    5TOO, 5TPQ

  • PubMed Abstract: 

    Members of enzyme superfamilies specialize in different reactions but often exhibit catalytic promiscuity for one another's reactions, consistent with catalytic promiscuity as an important driver in the evolution of new enzymes. Wanting to understand how catalytic promiscuity and other factors may influence evolution across a superfamily, we turned to the well-studied alkaline phosphatase (AP) superfamily, comparing three of its members, two evolutionarily distinct phosphatases and a phosphodiesterase. We mutated distinguishing active-site residues to generate enzymes that had a common Zn 2+ bimetallo core but little sequence similarity and different auxiliary domains. We then tested the catalytic capabilities of these pruned enzymes with a series of substrates. A substantial rate enhancement of ∼10 11 -fold for both phosphate mono- and diester hydrolysis by each enzyme indicated that the Zn 2+ bimetallo core is an effective mono/di-esterase generalist and that the bimetallo cores were not evolutionarily tuned to prefer their cognate reactions. In contrast, our pruned enzymes were ineffective sulfatases, and this limited promiscuity may have provided a driving force for founding the distinct one-metal-ion branch that contains all known AP superfamily sulfatases. Finally, our pruned enzymes exhibited 10 7 -10 8 -fold phosphotriesterase rate enhancements, despite absence of such enzymes within the AP superfamily. We speculate that the superfamily active-site architecture involved in nucleophile positioning prevents accommodation of the additional triester substituent. Overall, we suggest that catalytic promiscuity, and the ease or difficulty of remodeling and building onto existing protein scaffolds, have greatly influenced the course of enzyme evolution. Uncovering principles and properties of enzyme function, promiscuity, and repurposing provides lessons for engineering new enzymes.


  • Organizational Affiliation

    From the Department of Biochemistry, Beckman Center.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Alkaline phosphatase
A, B
446Escherichia coli K-12Mutation(s): 5 
Gene Names: phoAb0383JW0374
EC: 3.1.3.1
UniProt
Find proteins for P00634 (Escherichia coli (strain K12))
Explore P00634 
Go to UniProtKB:  P00634
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP00634
Sequence Annotations
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.45 Å
  • R-Value Free: 0.219 
  • R-Value Work: 0.165 
  • R-Value Observed: 0.167 
  • Space Group: P 63 2 2
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 160.72α = 90
b = 160.72β = 90
c = 138.36γ = 120
Software Package:
Software NamePurpose
BUSTERrefinement
XDSdata reduction
Aimlessdata scaling
PHASERphasing

Structure Validation

View Full Validation Report



Entry History & Funding Information

Deposition Data


Funding OrganizationLocationGrant Number
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)United StatesGM64798

Revision History  (Full details and data files)

  • Version 1.0: 2017-11-01
    Type: Initial release
  • Version 1.1: 2017-11-08
    Changes: Database references
  • Version 1.2: 2018-01-03
    Changes: Database references
  • Version 1.3: 2018-04-18
    Changes: Data collection
  • Version 1.4: 2020-01-01
    Changes: Author supporting evidence
  • Version 1.5: 2023-10-04
    Changes: Data collection, Database references, Derived calculations, Refinement description