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PDBsum entry 1khl

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Hydrolase PDB id
1khl
Jmol
Contents
Protein chains
444 a.a. *
Ligands
PO4 ×2
Metals
_ZN ×4
Waters ×307
* Residue conservation analysis

References listed in PDB file
Key reference
Title Artificial evolution of an enzyme active site: structural studies of three highly active mutants of escherichia coli alkaline phosphatase.
Authors M.H.Le du, C.Lamoure, B.H.Muller, O.V.Bulgakov, E.Lajeunesse, A.Ménez, J.C.Boulain.
Ref. J Mol Biol, 2002, 316, 941-953. [DOI no: 10.1006/jmbi.2001.5384]
PubMed id 11884134
Abstract
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.
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.
Secondary reference #1
Title Reaction mechanism of alkaline phosphatase based on crystal structures. Two-Metal ion catalysis.
Authors E.E.Kim, H.W.Wyckoff.
Ref. J Mol Biol, 1991, 218, 449-464.
PubMed id 2010919
Abstract
PROCHECK
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