1kh7
From Proteopedia
(New page: 200px<br /><applet load="1kh7" size="450" color="white" frame="true" align="right" spinBox="true" caption="1kh7, resolution 2.4Å" /> '''E. COLI ALKALINE PHOS...) |
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| - | [[Image:1kh7.jpg|left|200px]]<br /><applet load="1kh7" size=" | + | [[Image:1kh7.jpg|left|200px]]<br /><applet load="1kh7" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="1kh7, resolution 2.4Å" /> | caption="1kh7, resolution 2.4Å" /> | ||
'''E. COLI ALKALINE PHOSPHATASE MUTANT (D153GD330N)'''<br /> | '''E. COLI ALKALINE PHOSPHATASE MUTANT (D153GD330N)'''<br /> | ||
==Overview== | ==Overview== | ||
| - | The crystal structure of three mutants of Escherichia coli alkaline | + | 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. |
==About this Structure== | ==About this Structure== | ||
| - | 1KH7 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] with ZN, MG and SO4 as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Alkaline_phosphatase Alkaline phosphatase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.1.3.1 3.1.3.1] Full crystallographic information is available from [http:// | + | 1KH7 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] with <scene name='pdbligand=ZN:'>ZN</scene>, <scene name='pdbligand=MG:'>MG</scene> and <scene name='pdbligand=SO4:'>SO4</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Alkaline_phosphatase Alkaline phosphatase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.1.3.1 3.1.3.1] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1KH7 OCA]. |
==Reference== | ==Reference== | ||
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[[Category: Escherichia coli]] | [[Category: Escherichia coli]] | ||
[[Category: Single protein]] | [[Category: Single protein]] | ||
| - | [[Category: Bulgakov, O | + | [[Category: Bulgakov, O V.]] |
| - | [[Category: Du, M | + | [[Category: Du, M H.Le.]] |
[[Category: Laje, E.]] | [[Category: Laje, E.]] | ||
[[Category: Lamoure, C.]] | [[Category: Lamoure, C.]] | ||
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[[Category: hydrolase]] | [[Category: hydrolase]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 13:34:07 2008'' |
Revision as of 11:34, 21 February 2008
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E. COLI ALKALINE PHOSPHATASE MUTANT (D153GD330N)
Overview
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.
About this Structure
1KH7 is a Single protein structure of sequence from Escherichia coli with , and as ligands. Active as Alkaline phosphatase, with EC number 3.1.3.1 Full crystallographic information is available from OCA.
Reference
Artificial evolution of an enzyme active site: structural studies of three highly active mutants of Escherichia coli alkaline phosphatase., Le Du MH, Lamoure C, Muller BH, Bulgakov OV, Lajeunesse E, Menez A, Boulain JC, J Mol Biol. 2002 Mar 1;316(4):941-53. PMID:11884134
Page seeded by OCA on Thu Feb 21 13:34:07 2008
Categories: Alkaline phosphatase | Escherichia coli | Single protein | Bulgakov, O V. | Du, M H.Le. | Laje, E. | Lamoure, C. | Muller, B. H. | MG | SO4 | ZN | Hydrolase
