1mn7: Difference between revisions
New page: left|200px<br /><applet load="1mn7" size="450" color="white" frame="true" align="right" spinBox="true" caption="1mn7, resolution 2.15Å" /> '''NDP kinase mutant (H... |
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[[Image:1mn7.jpg|left|200px]]<br /><applet load="1mn7" size=" | [[Image:1mn7.jpg|left|200px]]<br /><applet load="1mn7" size="350" color="white" frame="true" align="right" spinBox="true" | ||
caption="1mn7, resolution 2.15Å" /> | caption="1mn7, resolution 2.15Å" /> | ||
'''NDP kinase mutant (H122G;N119S;F64W) in complex with aBAZTTP'''<br /> | '''NDP kinase mutant (H122G;N119S;F64W) in complex with aBAZTTP'''<br /> | ||
==Overview== | ==Overview== | ||
Antiviral nucleoside analog therapies rely on their incorporation by viral | Antiviral nucleoside analog therapies rely on their incorporation by viral DNA polymerases/reverse transcriptase leading to chain termination. The analogs (3'-deoxy-3'-azidothymidine (AZT), 2',3'-didehydro-2',3'-dideoxythymidine (d4T), and other dideoxynucleosides) are sequentially converted into triphosphate by cellular kinases of the nucleoside salvage pathway and are often poor substrates of these enzymes. Nucleoside diphosphate (NDP) kinase phosphorylates the diphosphate derivatives of the analogs with an efficiency some 10(4) lower than for its natural substrates. Kinetic and structural studies of Dictyostelium and human NDP kinases show that the sugar 3'-OH, absent from all antiviral analogs, is required for catalysis. To improve the catalytic efficiency of NDP kinase on the analogs, we engineered several mutants with a protein OH group replacing the sugar 3'-OH. The substitution of Asn-115 in Ser and Leu-55 in His results in an NDP kinase mutant with an enhanced ability to phosphorylate antiviral derivatives. Transfection of the mutant enzyme in Escherichia coli results in an increased sensitivity to AZT. An x-ray structure at 2.15-A resolution of the Dictyostelium enzyme bearing the serine substitution in complex with the R(p)-alpha-borano-triphosphate derivative of AZT shows that the enhanced activity reflects an improved geometry of binding and a favorable interaction of the 3'-azido group with the engineered serine. | ||
==About this Structure== | ==About this Structure== | ||
1MN7 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Dictyostelium_discoideum Dictyostelium discoideum] with MG and ABT as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Nucleoside-diphosphate_kinase Nucleoside-diphosphate kinase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.7.4.6 2.7.4.6] Full crystallographic information is available from [http:// | 1MN7 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Dictyostelium_discoideum Dictyostelium discoideum] with <scene name='pdbligand=MG:'>MG</scene> and <scene name='pdbligand=ABT:'>ABT</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Nucleoside-diphosphate_kinase Nucleoside-diphosphate kinase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.7.4.6 2.7.4.6] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1MN7 OCA]. | ||
==Reference== | ==Reference== | ||
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[[Category: ndp kinase-abazttp complex]] | [[Category: ndp kinase-abazttp complex]] | ||
''Page seeded by [http:// | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 13:56:56 2008'' | ||
Revision as of 14:56, 21 February 2008
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NDP kinase mutant (H122G;N119S;F64W) in complex with aBAZTTP
OverviewOverview
Antiviral nucleoside analog therapies rely on their incorporation by viral DNA polymerases/reverse transcriptase leading to chain termination. The analogs (3'-deoxy-3'-azidothymidine (AZT), 2',3'-didehydro-2',3'-dideoxythymidine (d4T), and other dideoxynucleosides) are sequentially converted into triphosphate by cellular kinases of the nucleoside salvage pathway and are often poor substrates of these enzymes. Nucleoside diphosphate (NDP) kinase phosphorylates the diphosphate derivatives of the analogs with an efficiency some 10(4) lower than for its natural substrates. Kinetic and structural studies of Dictyostelium and human NDP kinases show that the sugar 3'-OH, absent from all antiviral analogs, is required for catalysis. To improve the catalytic efficiency of NDP kinase on the analogs, we engineered several mutants with a protein OH group replacing the sugar 3'-OH. The substitution of Asn-115 in Ser and Leu-55 in His results in an NDP kinase mutant with an enhanced ability to phosphorylate antiviral derivatives. Transfection of the mutant enzyme in Escherichia coli results in an increased sensitivity to AZT. An x-ray structure at 2.15-A resolution of the Dictyostelium enzyme bearing the serine substitution in complex with the R(p)-alpha-borano-triphosphate derivative of AZT shows that the enhanced activity reflects an improved geometry of binding and a favorable interaction of the 3'-azido group with the engineered serine.
About this StructureAbout this Structure
1MN7 is a Single protein structure of sequence from Dictyostelium discoideum with and as ligands. Active as Nucleoside-diphosphate kinase, with EC number 2.7.4.6 Full crystallographic information is available from OCA.
ReferenceReference
Improving nucleoside diphosphate kinase for antiviral nucleotide analogs activation., Gallois-Montbrun S, Schneider B, Chen Y, Giacomoni-Fernandes V, Mulard L, Morera S, Janin J, Deville-Bonne D, Veron M, J Biol Chem. 2002 Oct 18;277(42):39953-9. Epub 2002 Aug 8. PMID:12171931
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