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PDBsum entry 1elx

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protein ligands metals Protein-protein interface(s) links
Hydrolase PDB id
1elx
Jmol
Contents
Protein chains
449 a.a. *
Ligands
PO4 ×2
Metals
_ZN ×4
_MG ×2
Waters ×605
* Residue conservation analysis
PDB id:
1elx
Name: Hydrolase
Title: E. Coli alkaline phosphatase mutant (s102a)
Structure: Alkaline phosphatase. Chain: a, b. Engineered: yes. Mutation: yes. Other_details: enzyme inhibited with phosphate
Source: Escherichia coli. Organism_taxid: 562. Strain: sm547. Gene: phoa. Expressed in: escherichia coli. Expression_system_taxid: 562.
Biol. unit: Dimer (from PDB file)
Resolution:
2.60Å     R-factor:   0.158     R-free:   0.207
Authors: B.Stec,M.Hehir,C.Brennan,M.Nolte,E.R.Kantrowitz
Key ref:
B.Stec et al. (1998). Kinetic and X-ray structural studies of three mutant E. coli alkaline phosphatases: insights into the catalytic mechanism without the nucleophile Ser102. J Mol Biol, 277, 647-662. PubMed id: 9533886 DOI: 10.1006/jmbi.1998.1635
Date:
10-Feb-98     Release date:   27-May-98    
PROCHECK
Go to PROCHECK summary
 Headers
 References

Protein chains
Pfam   ArchSchema ?
P00634  (PPB_ECOLI) -  Alkaline phosphatase
Seq:
Struc:
471 a.a.
449 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.3.1.3.1  - Alkaline phosphatase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: A phosphate monoester + H2O = an alcohol + phosphate
phosphate monoester
+ H(2)O
= alcohol
+
phosphate
Bound ligand (Het Group name = PO4)
corresponds exactly
      Cofactor: Mg(2+); Zn(2+)
Molecule diagrams generated from .mol files obtained from the KEGG ftp site
 Gene Ontology (GO) functional annotation 
  GO annot!
  Cellular component     periplasmic space   2 terms 
  Biological process     metabolic process   3 terms 
  Biochemical function     catalytic activity     10 terms  

 

 
    reference    
 
 
DOI no: 10.1006/jmbi.1998.1635 J Mol Biol 277:647-662 (1998)
PubMed id: 9533886  
 
 
Kinetic and X-ray structural studies of three mutant E. coli alkaline phosphatases: insights into the catalytic mechanism without the nucleophile Ser102.
B.Stec, M.J.Hehir, C.Brennan, M.Nolte, E.R.Kantrowitz.
 
  ABSTRACT  
 
Escherichia coli alkaline phosphatase (EC 3.1.3.1) is a non-specific phosphomonoesterase that catalyzes the hydrolysis reaction via a phosphoseryl intermediate to produce inorganic phosphate and the corresponding alcohol. We investigated the nature of the primary nucleophile, fulfilled by the deprotonated Ser102, in the catalytic mechanism by mutating this residue to glycine, alanine and cysteine. The efficiencies of the S102G, S102A and S102C enzymes were 6 x 10(5)-fold, 10(5)-fold and 10(4)-fold lower than the wild-type enzyme, respectively, as measured by the kcat/Km ratio, still substantially higher than the non-catalyzed reaction. In order to investigate the structural details of the altered active site, the enzymes were crystallized and their structures determined. The enzymes crystallized in a new crystal form corresponding to the space group P6322. Each structure has phosphate at each active site and shows little departure from the wild-type model. For the S102G and S102A enzymes, the phosphate occupies the same position as in the wild-type enzyme, while in the S102C enzyme it is displaced by 2.5 A. This kinetic and structural study suggests an explanation for differences in catalytic efficiency of the mutant enzymes and provides a means to study the nature and strength of different nucleophiles in the same environment. The analysis of these results provides insight into the mechanisms of other classes of phosphatases that do not utilize a serine nucleophile.
 
  Selected figure(s)  
 
Figure 2.
Figure 2. The active site of the wild-type E. coli alkaline phosphatase. Shown are Zn[1], Zn[2], Mg, phosphate (P[i]) and the side-chain ligands. Also shown is Ser102, which is phosphorylated during the reaction, and Arg166, which interacts with the phosphate.
Figure 4.
Figure 4. Stereoview of the (2F[o]−F[c]) electron density map (contoured at 1.3σ) of the active site in the S102A mutant structure. The electron density and the side-chain for one of the Zn[2]ligands (His370) are omitted for clarity.
 
  The above figures are reprinted by permission from Elsevier: J Mol Biol (1998, 277, 647-662) copyright 1998.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
20148233 J.T.Wang, Q.Xia, X.H.Zheng, H.Y.Chen, H.Chao, Z.W.Mao, and L.N.Ji (2010).
An effective approach to artificial nucleases using copper(II) complexes bearing nucleobases.
  Dalton Trans, 39, 2128-2136.  
20164409 N.J.Baxter, M.W.Bowler, T.Alizadeh, M.J.Cliff, A.M.Hounslow, B.Wu, D.B.Berkowitz, N.H.Williams, G.M.Blackburn, and J.P.Waltho (2010).
Atomic details of near-transition state conformers for enzyme phosphoryl transfer revealed by MgF-3 rather than by phosphoranes.
  Proc Natl Acad Sci U S A, 107, 4555-4560.
PDB codes: 2wf5 2whe
17951381 C.L.Naessan, W.Egge-Jacobsen, R.W.Heiniger, M.C.Wolfgang, F.E.Aas, A.Røhr, H.C.Winther-Larsen, and M.Koomey (2008).
Genetic and functional analyses of PptA, a phospho-form transferase targeting type IV pili in Neisseria gonorrhoeae.
  J Bacteriol, 190, 387-400.  
17008720 J.Wang, and E.R.Kantrowitz (2006).
Trapping the tetrahedral intermediate in the alkaline phosphatase reaction by substitution of the active site serine with threonine.
  Protein Sci, 15, 2395-2401.
PDB codes: 2g9y 2ga3
16041072 B.Stec, K.M.Holtz, C.L.Wojciechowski, and E.R.Kantrowitz (2005).
Structure of the wild-type TEM-1 beta-lactamase at 1.55 A and the mutant enzyme Ser70Ala at 2.1 A suggest the mode of noncovalent catalysis for the mutant enzyme.
  Acta Crystallogr D Biol Crystallogr, 61, 1072-1079.
PDB codes: 1zg4 1zg6
12707276 R.R.Boulanger, and E.R.Kantrowitz (2003).
Characterization of a monomeric Escherichia coli alkaline phosphatase formed upon a single amino acid substitution.
  J Biol Chem, 278, 23497-23501.  
11863460 P.J.O'Brien, and D.Herschlag (2002).
Alkaline phosphatase revisited: hydrolysis of alkyl phosphates.
  Biochemistry, 41, 3207-3225.  
11029583 B.Asgeirsson, J.B.Hauksson, and G.H.Gunnarsson (2000).
Dissociation and unfolding of cold-active alkaline phosphatase from atlantic cod in the presence of guanidinium chloride.
  Eur J Biochem, 267, 6403-6412.  
10747010 M.J.Jedrzejas, M.Chander, P.Setlow, and G.Krishnasamy (2000).
Structure and mechanism of action of a novel phosphoglycerate mutase from Bacillus stearothermophilus.
  EMBO J, 19, 1419-1431.
PDB code: 1ejj
  11055995 W.R.Silverman, C.Y.Tang, A.F.Mock, K.B.Huh, and D.M.Papazian (2000).
Mg(2+) modulates voltage-dependent activation in ether-à-go-go potassium channels by binding between transmembrane segments S2 and S3.
  J Gen Physiol, 116, 663-678.  
The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB codes are shown on the right.