Source:http://linkedlifedata.com/resource/pubmed/id/11123916
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| Predicate | Object |
|---|---|
| rdf:type | |
| lifeskim:mentions | |
| pubmed:issue |
51
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| pubmed:dateCreated |
2001-1-8
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| pubmed:abstractText |
The monomeric homing endonuclease PI-SceI harbors two catalytic centers which cooperate in the cleavage of the two strands of its extended recognition sequence. Structural and biochemical data suggest that catalytic center I contains Asp218, Asp229, and Lys403, while catalytic center II contains Asp326, Thr341, and Lys301. The analogy with I-CreI, for which the cocrystal structure with the DNA substrate has been determined, suggests that Asp218 and Asp229 in catalytic center I and Asp326 and Thr341 in catalytic center II serve as ligands for Mg(2+), the essential divalent metal ion cofactor which can be replaced by Mn(2+) in vitro. We have carried out a mutational analysis of these presumptive Mg(2+) ligands. The variants carrying an alanine or asparagine substitution bind DNA, but (with the exception of the D229N variant) are inactive in DNA cleavage in the presence of Mg(2+), demonstrating that these residues are important for cleavage. Our finding that the PI-SceI variants carrying single cysteine substitutions at these positions are inactive in the presence of the oxophilic Mg(2+) but active in the presence of the thiophilic Mn(2+) suggests that the amino acid residues at these positions are involved in cofactor binding. From the fact that in the presence of Mn(2+) the D218C and D326C variants are even more active than the wild-type enzyme, it is concluded that Asp218 and Asp326 are the principal Mg(2+) ligands of PI-SceI. On the basis of these findings and the available structural information, a model for the composition of the two Mg(2+) binding sites of PI-SceI is proposed.
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| pubmed:language |
eng
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| pubmed:journal | |
| pubmed:citationSubset |
IM
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| pubmed:chemical |
http://linkedlifedata.com/resource/pubmed/chemical/Aspartic Acid,
http://linkedlifedata.com/resource/pubmed/chemical/Cross-Linking Reagents,
http://linkedlifedata.com/resource/pubmed/chemical/DNA, Fungal,
http://linkedlifedata.com/resource/pubmed/chemical/DNA-Binding Proteins,
http://linkedlifedata.com/resource/pubmed/chemical/Endodeoxyribonucleases,
http://linkedlifedata.com/resource/pubmed/chemical/Ligands,
http://linkedlifedata.com/resource/pubmed/chemical/Magnesium,
http://linkedlifedata.com/resource/pubmed/chemical/Manganese,
http://linkedlifedata.com/resource/pubmed/chemical/Oligodeoxyribonucleotides,
http://linkedlifedata.com/resource/pubmed/chemical/Proton-Translocating ATPases,
http://linkedlifedata.com/resource/pubmed/chemical/Saccharomyces cerevisiae Proteins,
http://linkedlifedata.com/resource/pubmed/chemical/TFP1 protein, S cerevisiae
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| pubmed:status |
MEDLINE
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| pubmed:month |
Dec
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| pubmed:issn |
0006-2960
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| pubmed:author | |
| pubmed:issnType |
Print
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| pubmed:day |
26
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| pubmed:volume |
39
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| pubmed:owner |
NLM
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| pubmed:authorsComplete |
Y
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| pubmed:pagination |
15895-900
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| pubmed:dateRevised |
2006-11-15
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| pubmed:meshHeading |
pubmed-meshheading:11123916-Amino Acid Substitution,
pubmed-meshheading:11123916-Aspartic Acid,
pubmed-meshheading:11123916-Catalytic Domain,
pubmed-meshheading:11123916-Cross-Linking Reagents,
pubmed-meshheading:11123916-DNA, Fungal,
pubmed-meshheading:11123916-DNA-Binding Proteins,
pubmed-meshheading:11123916-Endodeoxyribonucleases,
pubmed-meshheading:11123916-Hydrolysis,
pubmed-meshheading:11123916-Ligands,
pubmed-meshheading:11123916-Magnesium,
pubmed-meshheading:11123916-Manganese,
pubmed-meshheading:11123916-Mutagenesis, Site-Directed,
pubmed-meshheading:11123916-Oligodeoxyribonucleotides,
pubmed-meshheading:11123916-Photochemistry,
pubmed-meshheading:11123916-Protein Binding,
pubmed-meshheading:11123916-Proton-Translocating ATPases,
pubmed-meshheading:11123916-Saccharomyces cerevisiae Proteins
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| pubmed:year |
2000
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| pubmed:articleTitle |
Identification of Asp218 and Asp326 as the principal Mg2+ binding ligands of the homing endonuclease PI-SceI.
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| pubmed:affiliation |
Institut für Biochemie, FB 08, Justus-Liebig-Universität, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany.
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| pubmed:publicationType |
Journal Article,
Comparative Study,
Research Support, Non-U.S. Gov't
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