6mht
From Proteopedia
(New page: 200px<br /><applet load="6mht" size="450" color="white" frame="true" align="right" spinBox="true" caption="6mht, resolution 2.05Å" /> '''TERNARY STRUCTURE OF...) |
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| - | [[Image:6mht.gif|left|200px]]<br /><applet load="6mht" size=" | + | [[Image:6mht.gif|left|200px]]<br /><applet load="6mht" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="6mht, resolution 2.05Å" /> | caption="6mht, resolution 2.05Å" /> | ||
'''TERNARY STRUCTURE OF HHAI METHYLTRANSFERASE WITH ADOHCY AND DNA CONTAINING 4'-THIO-2'DEOXYCYTIDINE AT THE TARGET'''<br /> | '''TERNARY STRUCTURE OF HHAI METHYLTRANSFERASE WITH ADOHCY AND DNA CONTAINING 4'-THIO-2'DEOXYCYTIDINE AT THE TARGET'''<br /> | ||
==Overview== | ==Overview== | ||
| - | 4'-Thio-2'-deoxycytidine was synthesized as a 5'- protected | + | 4'-Thio-2'-deoxycytidine was synthesized as a 5'- protected phosphoramidite compatible with solid phase DNA synthesis. When incorporated as the target cytosine (C*) in the GC*GC recognition sequence for the DNA methyltransferase M. HhaI, methyl transfer was strongly inhibited. In contrast, these same oligonucleotides were normal substrates for the cognate restriction endonuclease R. HhaI and its isoschizomer R. Hin P1I. M. HhaI was able to bind both 4'-thio-modified DNA and unmodified DNA to equivalent extents under equilibrium conditions. However, the presence of 4'-thio-2'-deoxycytidine decreased the half-life of the complex by >10-fold. The crystal structure of a ternary complex of M. HhaI, AdoMet and DNA containing 4'-thio-2'-deoxycytidine was solved at 2.05 A resolution with a crystallographic R-factor of 0.186 and R-free of 0.231. The structure is not grossly different from previously solved ternary complexes containing M. HhaI, DNA and AdoHcy. The difference electron density suggests partial methylation at C5 of the flipped target 4'-thio-2'-deoxycytidine. The inhibitory effect of the 4'sulfur atom on enzymatic activity may be traced to perturbation of a step in the methylation reaction after DNA binding but prior to methyl transfer. This inhibitory effect can be partially overcome after a considerably long time in the crystal environment where the packing prevents complex dissociation and the target is accurately positioned within the active site. |
==About this Structure== | ==About this Structure== | ||
| - | 6MHT is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Haemophilus_haemolyticus Haemophilus haemolyticus] with SAM as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Deleted_entry Deleted entry], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.1.1.73 2.1.1.73] Full crystallographic information is available from [http:// | + | 6MHT is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Haemophilus_haemolyticus Haemophilus haemolyticus] with <scene name='pdbligand=SAM:'>SAM</scene> as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Deleted_entry Deleted entry], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.1.1.73 2.1.1.73] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=6MHT OCA]. |
==Reference== | ==Reference== | ||
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[[Category: transferase]] | [[Category: transferase]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 19:16:34 2008'' |
Revision as of 17:16, 21 February 2008
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TERNARY STRUCTURE OF HHAI METHYLTRANSFERASE WITH ADOHCY AND DNA CONTAINING 4'-THIO-2'DEOXYCYTIDINE AT THE TARGET
Overview
4'-Thio-2'-deoxycytidine was synthesized as a 5'- protected phosphoramidite compatible with solid phase DNA synthesis. When incorporated as the target cytosine (C*) in the GC*GC recognition sequence for the DNA methyltransferase M. HhaI, methyl transfer was strongly inhibited. In contrast, these same oligonucleotides were normal substrates for the cognate restriction endonuclease R. HhaI and its isoschizomer R. Hin P1I. M. HhaI was able to bind both 4'-thio-modified DNA and unmodified DNA to equivalent extents under equilibrium conditions. However, the presence of 4'-thio-2'-deoxycytidine decreased the half-life of the complex by >10-fold. The crystal structure of a ternary complex of M. HhaI, AdoMet and DNA containing 4'-thio-2'-deoxycytidine was solved at 2.05 A resolution with a crystallographic R-factor of 0.186 and R-free of 0.231. The structure is not grossly different from previously solved ternary complexes containing M. HhaI, DNA and AdoHcy. The difference electron density suggests partial methylation at C5 of the flipped target 4'-thio-2'-deoxycytidine. The inhibitory effect of the 4'sulfur atom on enzymatic activity may be traced to perturbation of a step in the methylation reaction after DNA binding but prior to methyl transfer. This inhibitory effect can be partially overcome after a considerably long time in the crystal environment where the packing prevents complex dissociation and the target is accurately positioned within the active site.
About this Structure
6MHT is a Single protein structure of sequence from Haemophilus haemolyticus with as ligand. Active as Deleted entry, with EC number 2.1.1.73 Full crystallographic information is available from OCA.
Reference
DNA containing 4'-thio-2'-deoxycytidine inhibits methylation by HhaI methyltransferase., Kumar S, Horton JR, Jones GD, Walker RT, Roberts RJ, Cheng X, Nucleic Acids Res. 1997 Jul 15;25(14):2773-83. PMID:9207024
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