2AB4

Dissecting the Roles of a Strictly Conserved Tyrosine in Substrate Recognition and Catalysis by Pseudouridine 55 Synthase


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.40 Å
  • R-Value Free: 0.272 
  • R-Value Work: 0.222 

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This is version 1.4 of the entry. See complete history


Literature

Dissecting the roles of a strictly conserved tyrosine in substrate recognition and catalysis by pseudouridine 55 synthase.

Phannachet, K.Elias, Y.Huang, R.H.

(2005) Biochemistry 44: 15488-15494

  • DOI: https://doi.org/10.1021/bi050961w
  • Primary Citation of Related Structures:  
    2AB4

  • PubMed Abstract: 

    Sequence alignment of the TruA, TruB, RsuA, and RluA families of pseudouridine synthases (PsiS) identifies a strictly conserved aspartic acid, which has been shown to be the critical nucleophile for the PsiS-catalyzed formation of pseudouridine (Psi). However, superposition of the representative structures from these four families of enzymes identifies two additional amino acids, a lysine or an arginine (K/R) and a tyrosine (Y), from a K/RxY motif that are structurally conserved in the active site. We have created a series of Thermotoga maritima and Escherichia coli pseudouridine 55 synthase (Psi55S) mutants in which the conserved Y is mutated to other amino acids. A new crystal structure of the T. maritima Psi55S Y67F mutant in complex with a 5FU-RNA at 2.4 A resolution revealed formation of 5-fluoro-6-hydroxypseudouridine (5FhPsi), the same product previously seen in wild-type Psi55S-5FU-RNA complex structures. HPLC analysis confirmed efficient formation of 5FhPsi by both Psi55S Y67F and Y67L mutants but to a much lesser extent by the Y67A mutant when 5FU-RNA substrate was used. However, both HPLC analysis and a tritium release assay indicated that these mutants had no detectable enzymatic activity when the natural RNA substrate was used. The combined structural and mutational studies lead us to propose that the side chain of the conserved tyrosine in these four families of PsiS plays a dual role within the active site, maintaining the structural integrity of the active site through its hydrophobic phenyl ring and acting as a general base through its OH group for the proton abstraction required in the last step of PsiS-catalyzed formation of Psi.


  • Organizational Affiliation

    Department of Biochemistry, School of Molecular and Cellular Biology, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, Illinois 61801, USA.


Macromolecules

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Entity ID: 2
MoleculeChains Sequence LengthOrganismDetailsImage
tRNA pseudouridine synthase BB [auth A]309Thermotoga maritimaMutation(s): 1 
Gene Names: truB
EC: 5.4.99
UniProt
Find proteins for Q9WZW0 (Thermotoga maritima (strain ATCC 43589 / DSM 3109 / JCM 10099 / NBRC 100826 / MSB8))
Explore Q9WZW0 
Go to UniProtKB:  Q9WZW0
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupQ9WZW0
Sequence Annotations
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  • Reference Sequence

Find similar nucleic acids by:  Sequence   |   3D Structure  

Entity ID: 1
MoleculeChains LengthOrganismImage
5'-R(*CP*CP*AP*CP*GP*GP*UP*(FHU)P*CP*GP*AP*AP*UP*CP*CP*GP*UP*GP*GP*C)-3'A [auth B]20N/A
Sequence Annotations
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  • Reference Sequence
Small Molecules
Ligands 1 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
ZN
Query on ZN

Download Ideal Coordinates CCD File 
C [auth A]ZINC ION
Zn
PTFCDOFLOPIGGS-UHFFFAOYSA-N
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.40 Å
  • R-Value Free: 0.272 
  • R-Value Work: 0.222 
  • Space Group: P 21 21 2
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 157.87α = 90
b = 51.87β = 90
c = 57.15γ = 90
Software Package:
Software NamePurpose
DENZOdata reduction
SCALEPACKdata scaling
AMoREphasing
CNSrefinement

Structure Validation

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Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2005-12-13
    Type: Initial release
  • Version 1.1: 2008-04-30
    Changes: Version format compliance
  • Version 1.2: 2011-07-13
    Changes: Version format compliance
  • Version 1.3: 2021-10-20
    Changes: Database references, Derived calculations
  • Version 1.4: 2023-08-23
    Changes: Data collection, Refinement description