1MP4

W224H VARIANT OF S. ENTERICA RmlA


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.20 Å
  • R-Value Free: 0.288 
  • R-Value Work: 0.257 
  • R-Value Observed: 0.284 

wwPDB Validation   3D Report Full Report


This is version 1.5 of the entry. See complete history


Literature

Expanding pyrimidine diphosphosugar libraries via structure-based nucleotidylyltransferase engineering

Barton, W.A.Biggins, J.B.Jiang, J.Thorson, J.S.Nikolov, D.B.

(2002) Proc Natl Acad Sci U S A 99: 13397-13402

  • DOI: https://doi.org/10.1073/pnas.192468299
  • Primary Citation of Related Structures:  
    1MP3, 1MP4, 1MP5

  • PubMed Abstract: 

    In vitro "glycorandomization" is a chemoenzymatic approach for generating diverse libraries of glycosylated biomolecules based on natural product scaffolds. This technology makes use of engineered variants of specific enzymes affecting metabolite glycosylation, particularly nucleotidylyltransferases and glycosyltransferases. To expand the repertoire of UDP/dTDP sugars readily available for glycorandomization, we now report a structure-based engineering approach to increase the diversity of alpha-d-hexopyranosyl phosphates accepted by Salmonella enterica LT2 alpha-d-glucopyranosyl phosphate thymidylyltransferase (E(p)). This article highlights the design rationale, determined substrate specificity, and structural elucidation of three "designed" mutations, illustrating both the success and unexpected outcomes from this type of approach. In addition, a single amino acid substitution in the substrate-binding pocket (L89T) was found to significantly increase the set of alpha-d-hexopyranosyl phosphates accepted by E(p) to include alpha-d-allo-, alpha-d-altro-, and alpha-d-talopyranosyl phosphate. In aggregate, our results provide valuable blueprints for altering nucleotidylyltransferase specificity by design, which is the first step toward in vitro glycorandomization.


  • Organizational Affiliation

    Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
W224H Variant of S. Enterica RmlA Bound to UDP-Glucose
A, B
292Salmonella entericaMutation(s): 1 
EC: 2.7.7.24
UniProt
Find proteins for Q9F7G8 (Salmonella enterica)
Explore Q9F7G8 
Go to UniProtKB:  Q9F7G8
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupQ9F7G8
Sequence Annotations
Expand
  • Reference Sequence
Small Molecules
Ligands 1 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
UPG
Query on UPG

Download Ideal Coordinates CCD File 
C [auth A],
D [auth B]
URIDINE-5'-DIPHOSPHATE-GLUCOSE
C15 H24 N2 O17 P2
HSCJRCZFDFQWRP-JZMIEXBBSA-N
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.20 Å
  • R-Value Free: 0.288 
  • R-Value Work: 0.257 
  • R-Value Observed: 0.284 
  • Space Group: P 65 2 2
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 88.958α = 90
b = 88.958β = 90
c = 329.1γ = 120

Structure Validation

View Full Validation Report



Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2002-10-09
    Type: Initial release
  • Version 1.1: 2008-04-28
    Changes: Version format compliance
  • Version 1.2: 2011-07-13
    Changes: Version format compliance
  • Version 1.3: 2018-01-31
    Changes: Experimental preparation
  • Version 1.4: 2021-10-27
    Changes: Advisory, Database references, Derived calculations
  • Version 1.5: 2024-02-14
    Changes: Data collection, Refinement description