1A2N

STRUCTURE OF THE C115A MUTANT OF MURA COMPLEXED WITH THE FLUORINATED ANALOG OF THE REACTION TETRAHEDRAL INTERMEDIATE


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.80 Å
  • R-Value Work: 0.175 
  • R-Value Observed: 0.175 

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This is version 1.5 of the entry. See complete history


Literature

Stereochemical course of enzymatic enolpyruvyl transfer and catalytic conformation of the active site revealed by the crystal structure of the fluorinated analogue of the reaction tetrahedral intermediate bound to the active site of the C115A mutant of MurA

Skarzynski, T.Kim, D.H.Lees, W.J.Walsh, C.T.Duncan, K.

(1998) Biochemistry 37: 2572-2577

  • DOI: https://doi.org/10.1021/bi9722608
  • Primary Citation of Related Structures:  
    1A2N

  • PubMed Abstract: 

    MurA (UDP-GlcNAc enolpyruvyl transferase), the first enzyme in bacterial peptidoglycan biosynthesis, catalyzes the enolpyruvyl transfer from phosphoenolpyruvate (PEP) to the 3'-OH of UDP-GlcNAc by an addition-elimination mechanism that proceeds through a tetrahedral ketal intermediate. The crystal structure of the Cys115-to-Ala (C115A) mutant of Escherichia coli MurA complexed with a fluoro analogue of the tetrahedral intermediate revealed the absolute configuration of the adduct and the stereochemical course of the reaction. The fluorinated adduct was generated in a preincubation of wild-type MurA with (Z)-3-fluorophosphoenolpyruvate (FPEP) and UDP-GlcNAc and purified after enzyme denaturation. The fluorine substituent stabilizes the tetrahedral intermediate toward decomposition by a factor of 10(4)-10(6), facilitating manipulation of the adduct. The C115A mutant of MurA was utilized to avoid the microheterogeneity that arises in the wild-type MurA from the attack of Cys115 on C-2 of FPEP in competition with the formation of the fluorinated adduct. The crystal structure of the complex was determined to 2.8 A resolution, and the absolute configuration at C-2 of the adduct was found to be 2R. Thus, addition of the 3'-OH of UDP-GlcNAc is to the 2-si face of FPEP, corresponding to the 2-re face of PEP. Given the previous observation that, in D2O, the addition of D+ to C-3 of PEP proceeds from the 2-si face [Kim, D. H., Lees, W. J., and Walsh, C. T. (1995) J. Am. Chem. Soc. 117, 6380-6381], the addition across the double bond of PEP is anti. Also, because the overall stereochemical course has been shown to be either anti/syn or syn/anti [Lees, W. J., and Walsh, C. T. (1995) J. Am. Chem. Soc. 117, 7329-7337], it now follows that the stereochemistry of elimination of H+ from C-3 and Pi from C-2 of the tetrahedral intermediate of the reaction is syn.


  • Organizational Affiliation

    Glaxo Wellcome Research and Development, Medicines Research Centre, Stevenage, Hertfordshire, United Kingdom SG1 2NY. ts14913@ggr.co.uk


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
UDP-N-ACETYLGLUCOSAMINE ENOLPYRUVYL TRANSFERASE419Escherichia coliMutation(s): 1 
EC: 2.5.1.7
UniProt
Find proteins for P0A749 (Escherichia coli (strain K12))
Explore P0A749 
Go to UniProtKB:  P0A749
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP0A749
Sequence Annotations
Expand
  • Reference Sequence
Small Molecules
Ligands 1 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
TET
Query on TET

Download Ideal Coordinates CCD File 
B [auth A]URIDINE-DIPHOSPHATE-2(N-ACETYLGLUCOSAMINYL-3-FLUORO-2-PHOSPHONOOXY)PROPIONIC ACID
C20 H31 F N3 O23 P3
FUDVRUWZNPQAFR-BVDGEXFOSA-N
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.80 Å
  • R-Value Work: 0.175 
  • R-Value Observed: 0.175 
  • Space Group: P 3 2 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 111.15α = 90
b = 111.15β = 90
c = 67.51γ = 120
Software Package:
Software NamePurpose
X-PLORmodel building
PROLSQrefinement
X-PLORrefinement
MOSFLMdata reduction
CCP4data scaling
X-PLORphasing

Structure Validation

View Full Validation Report



Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 1998-04-29
    Type: Initial release
  • Version 1.1: 2008-03-24
    Changes: Version format compliance
  • Version 1.2: 2011-07-13
    Changes: Version format compliance
  • Version 1.3: 2019-08-14
    Changes: Data collection, Other
  • Version 1.4: 2021-11-03
    Changes: Database references, Derived calculations
  • Version 1.5: 2023-08-02
    Changes: Refinement description