1xo1
From Proteopedia
(New page: 200px<br /><applet load="1xo1" size="450" color="white" frame="true" align="right" spinBox="true" caption="1xo1, resolution 2.5Å" /> '''T5 5'-EXONUCLEASE MUT...) |
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| - | [[Image:1xo1.jpg|left|200px]]<br /><applet load="1xo1" size=" | + | [[Image:1xo1.jpg|left|200px]]<br /><applet load="1xo1" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="1xo1, resolution 2.5Å" /> | caption="1xo1, resolution 2.5Å" /> | ||
'''T5 5'-EXONUCLEASE MUTANT K83A'''<br /> | '''T5 5'-EXONUCLEASE MUTANT K83A'''<br /> | ||
==Overview== | ==Overview== | ||
| - | Efficient cellular DNA replication requires the activity of a 5'-3' | + | Efficient cellular DNA replication requires the activity of a 5'-3' exonuclease. These enzymes are able to hydrolyze DNA.DNA and RNA.DNA substrates exonucleolytically, and they are structure-specific endonucleases. The 5'-3' exonucleases are conserved in organisms as diverse as bacteriophage and mammals. Crystal structures of three representative enzymes identify two divalent-metal-binding sites typically separated by 8-10 A. Site-directed mutagenesis was used to investigate the roles of three lysine residues (K83, K196, and K215) situated near two metal-binding sites in bacteriophage T5 5'-3' exonuclease. Neither K196 nor K215 was essential for either the exo- or the endonuclease activity, but mutation of these residues increased the dissociation constant for the substrate from 5 nM to 200 nM (K196A) and 50 nM (K215A). Biochemical analysis demonstrated that K83 is absolutely required for exonucleolytic activity on single-stranded DNA but is not required for endonucleolytic cleavage of flap structures. Structural analysis of this mutant by x-ray crystallography showed no significant perturbations around the metal-binding sites in the active site. The wild-type protein has different pH optima for endonuclease and exonuclease activities. Taken together, these results suggest that different mechanisms for endo- and exonucleolytic hydrolysis are used by this multifunctional enzyme. |
==About this Structure== | ==About this Structure== | ||
| - | 1XO1 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Pseudomonas_phage_d3112 Pseudomonas phage d3112]. Active as [http://en.wikipedia.org/wiki/Exodeoxyribonuclease_(lambda-induced) Exodeoxyribonuclease (lambda-induced)], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.1.11.3 3.1.11.3] Full crystallographic information is available from [http:// | + | 1XO1 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Pseudomonas_phage_d3112 Pseudomonas phage d3112]. Active as [http://en.wikipedia.org/wiki/Exodeoxyribonuclease_(lambda-induced) Exodeoxyribonuclease (lambda-induced)], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.1.11.3 3.1.11.3] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1XO1 OCA]. |
==Reference== | ==Reference== | ||
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[[Category: Pseudomonas phage d3112]] | [[Category: Pseudomonas phage d3112]] | ||
[[Category: Single protein]] | [[Category: Single protein]] | ||
| - | [[Category: Ceska, T | + | [[Category: Ceska, T A.]] |
| - | [[Category: Sayers, J | + | [[Category: Sayers, J R.]] |
[[Category: Suck, D.]] | [[Category: Suck, D.]] | ||
[[Category: exonuclease]] | [[Category: exonuclease]] | ||
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[[Category: nuclease]] | [[Category: nuclease]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 15:56:51 2008'' |
Revision as of 13:56, 21 February 2008
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T5 5'-EXONUCLEASE MUTANT K83A
Overview
Efficient cellular DNA replication requires the activity of a 5'-3' exonuclease. These enzymes are able to hydrolyze DNA.DNA and RNA.DNA substrates exonucleolytically, and they are structure-specific endonucleases. The 5'-3' exonucleases are conserved in organisms as diverse as bacteriophage and mammals. Crystal structures of three representative enzymes identify two divalent-metal-binding sites typically separated by 8-10 A. Site-directed mutagenesis was used to investigate the roles of three lysine residues (K83, K196, and K215) situated near two metal-binding sites in bacteriophage T5 5'-3' exonuclease. Neither K196 nor K215 was essential for either the exo- or the endonuclease activity, but mutation of these residues increased the dissociation constant for the substrate from 5 nM to 200 nM (K196A) and 50 nM (K215A). Biochemical analysis demonstrated that K83 is absolutely required for exonucleolytic activity on single-stranded DNA but is not required for endonucleolytic cleavage of flap structures. Structural analysis of this mutant by x-ray crystallography showed no significant perturbations around the metal-binding sites in the active site. The wild-type protein has different pH optima for endonuclease and exonuclease activities. Taken together, these results suggest that different mechanisms for endo- and exonucleolytic hydrolysis are used by this multifunctional enzyme.
About this Structure
1XO1 is a Single protein structure of sequence from Pseudomonas phage d3112. Active as Exodeoxyribonuclease (lambda-induced), with EC number 3.1.11.3 Full crystallographic information is available from OCA.
Reference
Mutagenesis of conserved lysine residues in bacteriophage T5 5'-3' exonuclease suggests separate mechanisms of endo-and exonucleolytic cleavage., Garforth SJ, Ceska TA, Suck D, Sayers JR, Proc Natl Acad Sci U S A. 1999 Jan 5;96(1):38-43. PMID:9874768
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