PDBsum entry 1khj

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Hydrolase PDB id
Jmol PyMol
Protein chains
444 a.a. *
AF3 ×2
_ZN ×4
Waters ×439
* Residue conservation analysis
PDB id:
Name: Hydrolase
Title: E. Coli alkaline phosphatase mutant (d153hd330n) mimic of th transition states with aluminium fluoride
Structure: Alkaline phosphatase. Chain: a, b. Engineered: yes. Mutation: yes
Source: Escherichia coli. Organism_taxid: 562. Strain: wcc118. Expressed in: escherichia coli. Expression_system_taxid: 562.
Biol. unit: Dimer (from PQS)
2.30Å     R-factor:   0.180     R-free:   0.223
Authors: M.H.Le Du,C.Lamoure,B.H.Muller,O.V.Bulgakov,E.Lajeunesse,A.M J.C.Boulain
Key ref:
M.H.Le Du et al. (2002). Artificial evolution of an enzyme active site: structural studies of three highly active mutants of Escherichia coli alkaline phosphatase. J Mol Biol, 316, 941-953. PubMed id: 11884134 DOI: 10.1006/jmbi.2001.5384
30-Nov-01     Release date:   13-Mar-02    
Go to PROCHECK summary

Protein chains
Pfam   ArchSchema ?
P00634  (PPB_ECOLI) -  Alkaline phosphatase
471 a.a.
444 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 7 residue positions (black crosses)

 Enzyme reactions 
   Enzyme class: E.C.  - Alkaline phosphatase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: A phosphate monoester + H2O = an alcohol + phosphate
phosphate monoester
+ H(2)O
= alcohol
+ phosphate
      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   4 terms 
  Biochemical function     catalytic activity     11 terms  


DOI no: 10.1006/jmbi.2001.5384 J Mol Biol 316:941-953 (2002)
PubMed id: 11884134  
Artificial evolution of an enzyme active site: structural studies of three highly active mutants of Escherichia coli alkaline phosphatase.
M.H.Le Du, C.Lamoure, B.H.Muller, O.V.Bulgakov, E.Lajeunesse, A.Ménez, J.C.Boulain.
The crystal structure of three mutants of Escherichia coli alkaline phosphatase with catalytic activity (k(cat)) enhancement as compare to the wild-type enzyme is described in different states. The biological aspects of this study have been reported elsewhere. The structure of the first mutant, D330N, which is threefold more active than the wild-type enzyme, was determined with phosphate in the active site, or with aluminium fluoride, which mimics the transition state. These structures reveal, in particular, that this first mutation does not alter the active site. The second mutant, D153H-D330N, is 17-fold more active than the wild-type enzyme and activated by magnesium, but its activity drops after few days. The structure of this mutant was solved under four different conditions. The phosphate-free enzyme was studied in an inactivated form with zinc at site M3, or after activation by magnesium. The comparison of these two forms free of phosphate illustrates the mechanism of the magnesium activation of the catalytic serine residue. In the presence of magnesium, the structure was determined with phosphate, or aluminium fluoride. The drop in activity of the mutant D153H-D330N could be explained by the instability of the metal ion at M3. The analysis of this mutant helped in the design of the third mutant, D153G-D330N. This mutant is up to 40-fold more active than the wild-type enzyme, with a restored robustness of the enzyme stability. The structure is presented here with covalently bound phosphate in the active site, representing the first phosphoseryl intermediate of a highly active alkaline phosphatase. This study shows how structural analysis may help to progress in the improvement of an enzyme catalytic activity (k(cat)), and explains the structural events associated with this artificial evolution.
  Selected figure(s)  
Figure 2.
Figure 2. (a) Ball and stick representation in stereo view of Asp51, Ser102, Zn2 and Zn3 in the active site of APD153HD330N(Zn), and 1s contoured 2Fo - Fc map. (b) Ball and stick representation in stereo view of Asp51, Ser102, Zn2 and Mg3 in the active site of APD153HD330N(Mg), and 1s contoured 2Fo - Fc map.
Figure 4.
Figure 4. (a) Ribbon, and ball and stick representation of the active site of APD153GD330N and of the interactions that involve resi- dues Asp51, Ser102, Gly153, Thr155, Arg166, Glu322, Lys328, water molecules Wat1 to Wat4, and Zn1, Zn2 and Mg3, in the presence of phosphate in the active site. (b) Ball and stick representation in stereo view of the active site of the phosphoseryl intermediate of APD153GD330N, and 1s contoured 2Fo - Fc map.
  The above figures are reprinted by permission from Elsevier: J Mol Biol (2002, 316, 941-953) copyright 2002.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
22785315 T.Nakamura, Y.Zhao, Y.Yamagata, Y.J.Hua, and W.Yang (2012).
Watching DNA polymerase η make a phosphodiester bond.
  Nature, 487, 196-201.
PDB codes: 4ecq 4ecr 4ecs 4ect 4ecu 4ecv 4ecw 4ecx 4ecy 4ecz 4ed0 4ed1 4ed2 4ed3 4ed6 4ed7 4ed8
  19916164 D.Koutsioulis, A.Lyskowski, S.Mäki, E.Guthrie, G.Feller, V.Bouriotis, and P.Heikinheimo (2010).
Coordination sphere of the third metal site is essential to the activity and metal selectivity of alkaline phosphatases.
  Protein Sci, 19, 75-84.
PDB codes: 2w5v 2w5w 2w5x
18851975 J.G.Zalatan, T.D.Fenn, and D.Herschlag (2008).
Comparative enzymology in the alkaline phosphatase superfamily to determine the catalytic role of an active-site metal ion.
  J Mol Biol, 384, 1174-1189.
PDB code: 3dyc
17488874 A.T.Torelli, J.Krucinska, and J.E.Wedekind (2007).
A comparison of vanadate to a 2'-5' linkage at the active site of a small ribozyme suggests a role for water in transition-state stabilization.
  RNA, 13, 1052-1070.
PDB codes: 2p7d 2p7e 2p7f
17156422 C.D.Martin, G.Rojas, J.N.Mitchell, K.J.Vincent, J.Wu, J.McCafferty, and D.J.Schofield (2006).
A simple vector system to improve performance and utilisation of recombinant antibodies.
  BMC Biotechnol, 6, 46.  
16815919 P.Llinas, M.Masella, T.Stigbrand, A.Ménez, E.A.Stura, and M.H.Le Du (2006).
Structural studies of human alkaline phosphatase in complex with strontium: implication for its secondary effect in bones.
  Protein Sci, 15, 1691-1700.
PDB code: 2glq
12948780 P.A.Dalby (2003).
Optimising enzyme function by directed evolution.
  Curr Opin Struct Biol, 13, 500-505.  
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