8Y1Q

Crystal structure of Aquifex aeolicus dUTPase complexed with dUMP.


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.42 Å
  • R-Value Free: 0.273 
  • R-Value Work: 0.233 
  • R-Value Observed: 0.237 

Starting Model: in silico
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Literature

dUTP pyrophosphatases from hyperthermophilic eubacterium and archaeon: Structural and functional examinations on the suitability for PCR application.

Fukui, K.Kondo, N.Murakawa, T.Baba, S.Kumasaka, T.Yano, T.

(2024) Protein Sci 33: e5185-e5185

  • DOI: https://doi.org/10.1002/pro.5185
  • Primary Citation of Related Structures:  
    8Y1Q, 8Y1W

  • PubMed Abstract: 

    Deoxyuridine triphosphate pyrophosphatase (DUT) suppresses incorporation of uracil into genomic DNA during replication. Thermostable DUTs from hyperthermophilic archaea such as Thermococcus pacificus enhance PCR amplification by preventing misincorporation of dUTP generated by spontaneous deamination of dCTP. However, it is necessary to elucidate whether DUTs do not cause dNTP imbalances during PCR by unwanted side activity. Moreover, it has been unknown what structural features define the thermostability of those DUTs. Here, DUT from a hyperthermophilic eubacterium, Aquifex aeolicus (Aa-DUT), was characterized together with those from T. pacificus (Tp-DUT). Aa-DUT was as thermostable as Tp-DUT up to at least 95°C. The crystal structures of the two thermostable enzymes were determined, which revealed that the structures of Aa-DUT and Tp-DUT resembled those of monofunctional and bifunctional DUTs, respectively. Generally, bifunctional DUTs harbor the dCTP deaminase activity in addition to the DUT activity. However, not only Aa-DUT but also Tp-DUT showed poor activity towards dCTP, indicating both enzymes are monofunctional. We further examined eight types of parameters related to thermostability of protein structure and found that the thermostability of Aa-DUT and Tp-DUT might be accomplished by increased numbers of ion pairs on the protein surface. Finally, we verified that Aa-DUT promoted PCR amplification with Pfu DNA polymerase to the same extent as Tp-DUT. Collectively, we conclude that both DUTs from hyperthermophiles maintain the enzymatic activity at high temperatures without consuming dCTP due to the lack of the deaminate activity.


  • Organizational Affiliation

    Department of Biochemistry, Faculty of Medicine, Osaka Medical and Pharmaceutical University, Takatsuki, Osaka, Japan.


Macromolecules
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Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Deoxyuridine 5'-triphosphate nucleotidohydrolase
A, B, C
150Aquifex aeolicusMutation(s): 0 
Gene Names: dutaq_220
EC: 3.6.1.23
UniProt
Find proteins for O66592 (Aquifex aeolicus (strain VF5))
Explore O66592 
Go to UniProtKB:  O66592
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupO66592
Sequence Annotations
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.42 Å
  • R-Value Free: 0.273 
  • R-Value Work: 0.233 
  • R-Value Observed: 0.237 
  • Space Group: P 41
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 61.26α = 90
b = 61.26β = 90
c = 112.66γ = 90
Software Package:
Software NamePurpose
XDSdata reduction
PHENIXrefinement
Cootmodel building
PHENIXphasing
XDSdata scaling

Structure Validation

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Ligand Structure Quality Assessment 


Entry History & Funding Information

Deposition Data


Funding OrganizationLocationGrant Number
Not funded--

Revision History  (Full details and data files)

  • Version 1.0: 2024-11-06
    Type: Initial release