7OVG

The C146A variant of an amidase from Pyrococcus horikoshii with bound acetamide


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.65 Å
  • R-Value Free: 0.182 
  • R-Value Work: 0.151 
  • R-Value Observed: 0.153 

Starting Model: experimental
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Ligand Structure Quality Assessment 


This is version 1.2 of the entry. See complete history

Re-refinement Note

This entry reflects an alternative modeling of the original data in: 6YPA


Literature

The structures of the C146A variant of the amidase from Pyrococcus horikoshii bound to glutaramide and acetamide suggest the basis of amide recognition.

Makumire, S.Su, S.Weber, B.W.Woodward, J.D.Wangari Kimani, S.Hunter, R.Sewell, B.T.

(2022) J Struct Biol 214: 107859-107859

  • DOI: https://doi.org/10.1016/j.jsb.2022.107859
  • Primary Citation of Related Structures:  
    7OVG

  • PubMed Abstract: 

    The nitrilase superfamily enzymes from Pyrococcus abyssi and Pyrococcus horikoshii hydrolyze several different amides. No nitriles that we tested were hydrolyzed by either enzyme. Propionamide and acetamide were the most rapidly hydrolyzed of all the substrates tested. Amide substrate docking studies on the wild-type and C146A variant P. horikoshii enzymes suggest a sequence in which the incoming amide substrate initially hydrogen bonds to the amino group of Lys-113 and the backbone carbonyl of Asn-171. When steric hindrance is relieved by replacing the cysteine with alanine, the amide then docks such that the amino group of Lys-113 and the backbone amide of Phe-147 are hydrogen-bonded to the substrate carbonyl oxygen, while the backbone carbonyl oxygen of Asn-171 and the carboxyl oxygen of Glu-42 are hydrogen-bonded to the amino group of the substrate. Here, we confirm the location of the acetamide and glutaramide ligands experimentally in well-resolved crystal structures of the C146A mutant of the enzyme from P. horikoshii. This ligand location suggests that there is no direct interaction between the substrate amide and the other active site glutamate, Glu-120, and supports an active-site geometry leading to the formation of the thioester intermediate via an attack on the si-face of the amide by the sulfhydryl of the active site cysteine.


  • Organizational Affiliation

    Structural Biology Research Unit, Department of Integrative Biomedical Sciences, University of Cape Town, Observatory 7925, South Africa. Electronic address: [email protected].


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
CN hydrolase domain-containing protein
A, B
263Pyrococcus horikoshii OT3Mutation(s): 1 
Gene Names: PH0642
UniProt
Find proteins for O58376 (Pyrococcus horikoshii (strain ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3))
Explore O58376 
Go to UniProtKB:  O58376
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupO58376
Sequence Annotations
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.65 Å
  • R-Value Free: 0.182 
  • R-Value Work: 0.151 
  • R-Value Observed: 0.153 
  • Space Group: P 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 44.979α = 68.09
b = 57.012β = 85.16
c = 61.358γ = 76.93
Software Package:
Software NamePurpose
REFMACrefinement
XDSdata reduction
XDSdata scaling
PHENIXphasing

Structure Validation

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Ligand Structure Quality Assessment 


Entry History & Funding Information

Deposition Data


Funding OrganizationLocationGrant Number
Global Challenges Research FundUnited KingdomST/R002754/1

Revision History  (Full details and data files)

  • Version 1.0: 2021-07-21
    Type: Initial release
  • Version 1.1: 2022-05-04
    Changes: Database references
  • Version 1.2: 2024-01-31
    Changes: Data collection, Refinement description