Abstract
A novel mechanism of DNA endonucleolytic cleavage has been visualized for the homing endonuclease I-PpoI by trapping the uncleaved enzyme-substrate complex and comparing it to the previously visualized product complex. This enzyme employs a unique single metal mechanism. A magnesium ion is coordinated by an asparagine residue and two DNA oxygen atoms and stabilizes the phosphoanion transition state and the 3'oxygen leaving group. A hydrolytic water molecule is activated by a histidine residue for an in-line attack on the scissile phosphate. A strained enzyme-substrate-metal complex is formed before cleavage, then relaxed during the reaction.
MeSH terms
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Amino Acid Substitution / genetics
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Animals
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Binding Sites
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Catalysis
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Cations / metabolism
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Crystallography, X-Ray
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DNA / chemistry
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DNA / genetics
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DNA / metabolism
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Electrons
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Endodeoxyribonucleases / chemistry*
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Endodeoxyribonucleases / genetics
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Endodeoxyribonucleases / metabolism*
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Fourier Analysis
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Magnesium / metabolism
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Models, Chemical
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Models, Molecular
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Molecular Sequence Data
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Oxygen / metabolism
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Phosphates / metabolism
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Physarum polycephalum / enzymology*
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Protein Conformation
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Sodium / metabolism
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Solvents
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Structure-Activity Relationship
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Water / chemistry
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Water / metabolism
Substances
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Cations
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Phosphates
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Solvents
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Water
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DNA
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Sodium
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Endodeoxyribonucleases
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I-Ppo endonuclease
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Magnesium
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Oxygen
Associated data
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PDB/1A73
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PDB/1CYQ
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PDB/1CZ0