PDBsum entry 2nwr

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Transferase PDB id
Protein chains
257 a.a. *
PO4 ×2
PEP ×2
Waters ×439
* Residue conservation analysis
PDB id:
Name: Transferase
Title: Crystal structure of c11n mutant of kdo8p synthase in comple
Structure: 2-dehydro-3-deoxyphosphooctonate aldolase. Chain: a, b. Synonym: phospho-2-dehydro-3-deoxyoctonate aldolase, 3-deox octulosonic acid 8-phosphate synthetase, kdo-8-phosphate sy kdo 8-p synthase, kdops. Engineered: yes. Mutation: yes
Source: Aquifex aeolicus. Organism_taxid: 63363. Gene: kdsa. Expressed in: escherichia coli. Expression_system_taxid: 562
1.50Å     R-factor:   0.227     R-free:   0.247
Authors: F.Kona,X.Xu,P.Martin,P.Kuzmic,D.L.Gatti
Key ref: F.Kona et al. (2007). Structural and mechanistic changes along an engineered path from metallo to nonmetallo 3-deoxy-D-manno-octulosonate 8-phosphate synthases. Biochemistry, 46, 4532-4544. PubMed id: 17381075 DOI: 10.1021/bi6024879
16-Nov-06     Release date:   24-Apr-07    
Go to PROCHECK summary

Protein chains
Pfam   ArchSchema ?
O66496  (KDSA_AQUAE) -  2-dehydro-3-deoxyphosphooctonate aldolase
267 a.a.
257 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 1 residue position (black cross)

 Enzyme reactions 
   Enzyme class: E.C.  - 3-deoxy-8-phosphooctulonate synthase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: Phosphoenolpyruvate + D-arabinose 5-phosphate + H2O = 2-dehydro-3- deoxy-D-octonate 8-phosphate + phosphate
Bound ligand (Het Group name = PEP)
corresponds exactly
+ D-arabinose 5-phosphate
+ H(2)O
= 2-dehydro-3- deoxy-D-octonate 8-phosphate
Bound ligand (Het Group name = PO4)
corresponds exactly
Molecule diagrams generated from .mol files obtained from the KEGG ftp site
 Gene Ontology (GO) functional annotation 
  GO annot!
  Cellular component     cytoplasm   1 term 
  Biological process     metabolic process   4 terms 
  Biochemical function     catalytic activity     3 terms  


DOI no: 10.1021/bi6024879 Biochemistry 46:4532-4544 (2007)
PubMed id: 17381075  
Structural and mechanistic changes along an engineered path from metallo to nonmetallo 3-deoxy-D-manno-octulosonate 8-phosphate synthases.
F.Kona, X.Xu, P.Martin, P.Kuzmic, D.L.Gatti.
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.

Literature references that cite this PDB file's key reference

  PubMed id Reference
20533322 A.Roberts, C.Furdui, and K.S.Anderson (2010).
Observation of a chemically labile, noncovalent enzyme intermediate in the reaction of metal-dependent Aquifex pyrophilus KDO8PS by time-resolved mass spectrometry.
  Rapid Commun Mass Spectrom, 24, 1919-1924.  
19622749 P.Tumbale, and K.Brew (2009).
Characterization of a metal-independent CAZy family 6 glycosyltransferase from Bacteroides ovatus.
  J Biol Chem, 284, 25126-25134.  
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