2NWS

Structural and mechanistic changes along an engineered path from metallo to non-metallo KDO8P synthase


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.80 Å
  • R-Value Free: 0.268 
  • R-Value Work: 0.234 
  • R-Value Observed: 0.234 

Starting Model: experimental
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This is version 1.4 of the entry. See complete history


Literature

Structural and Mechanistic Changes along an Engineered Path from Metallo to Nonmetallo 3-Deoxy-d-manno-octulosonate 8-Phosphate Synthases.

Kona, F.Xu, X.Martin, P.Kuzmic, P.Gatti, D.L.

(2007) Biochemistry 46: 4532-4544

  • DOI: https://doi.org/10.1021/bi6024879

  • PubMed Abstract: 

    There are two classes of KDO8P synthases characterized respectively by the presence or absence of a metal in the active site. The nonmetallo KDO8PS from Escherichia coli and the metallo KDO8PS from Aquifex aeolicus are the best characterized members of each class. All amino acid residues that make important contacts with the substrates are conserved in both enzymes with the exception of Pro-10, Cys-11, Ser-235, and Gln-237 of the A. aeolicus enzyme, which correspond respectively to Met-25, Asn-26, Pro-252, and Ala-254 in the E. coli enzyme. Interconversion between the two forms of KDO8P synthases can be achieved by substituting the metal-coordinating cysteine of metallo synthases with the corresponding asparagine of nonmetallo synthases, and vice versa. In this report we describe the structural changes elicited by the C11N mutation and by three combinations of mutations (P10M/C11N, C11N/S235P/Q237A, and P10M/C11N/S235P/Q237A) situated along possible evolutionary paths connecting the A. aeolicus and the E. coli enzyme. All four mutants are not capable of binding metal and lack the structural asymmetry among subunits with regard to substrate binding and conformation of the L7 loop, which is typical of A. aeolicus wild-type KDO8PS but is absent in the E. coli enzyme. Despite the lack of the active site metal, the mutant enzymes display levels of activity ranging from 46% to 24% of the wild type. With the sole exception of the quadruple mutant, metal loss does not affect the thermal stability of KDO8PS. The free energy of unfolding in water is also either unchanged or even increased in the mutant enzymes, suggesting that the primary role of the active site metal in A. aeolicus KDO8PS is not to increase the enzyme stability. In all four mutants A5P binding displaces a water molecule located on the si side of PEP. In particular, in the double and triple mutant, A5P binds with the aldehyde carbonyl in hydrogen bond distance of Asn-11, while in the wild type this functional group points away from Cys-11. This alternative conformation of A5P is likely to have functional significance as it resembles the conformation of the acyclic reaction intermediate, which is observed here for the first time in some of the active sites of the triple mutant. The direct visualization of this intermediate by X-ray crystallography confirms earlier mechanistic models of KDO8P synthesis. In particular, the configuration of the C2 chiral center of the intermediate supports a model of the reaction in nonmetallo KDO8PS, in which water attacks an oxocarbenium ion or PEP from the si side of C2. Several explanations are offered to reconcile this observation with the fact that no water molecule is observed at this position in the mutant enzymes in the presence of both PEP and A5P. Significant differences were observed between the wild-type and the mutant enzymes in the Km values for PEP and A5P and in the Kd values for inorganic phosphate and R5P. These differences may reflect an evolutionary adaptation of metallo and nonmetallo KDO8PS's to the cellular concentrations of these metabolites in their respective hosts.


  • Organizational Affiliation

    Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
2-dehydro-3-deoxyphosphooctonate aldolase
A, B
267Aquifex aeolicusMutation(s): 1 
Gene Names: kdsA
EC: 2.5.1.55
UniProt
Find proteins for O66496 (Aquifex aeolicus (strain VF5))
Explore O66496 
Go to UniProtKB:  O66496
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupO66496
Sequence Annotations
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.80 Å
  • R-Value Free: 0.268 
  • R-Value Work: 0.234 
  • R-Value Observed: 0.234 
  • Space Group: P 31 2 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 84.466α = 90
b = 84.466β = 90
c = 159.907γ = 120
Software Package:
Software NamePurpose
CNSrefinement

Structure Validation

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Ligand Structure Quality Assessment 


Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2007-04-24
    Type: Initial release
  • Version 1.1: 2008-05-01
    Changes: Version format compliance
  • Version 1.2: 2011-07-13
    Changes: Derived calculations, Version format compliance
  • Version 1.3: 2021-10-20
    Changes: Database references, Derived calculations
  • Version 1.4: 2023-08-30
    Changes: Data collection, Refinement description