Structural and Kinetic Characterization of Thymidine Kinase from Leishmania major.
Leishmania spp. is a protozoan parasite and the causative agent of leishmaniasis. Thymidine kinase (TK) catalyses the transfer of the γ-phosphate of ATP to 2'-deoxythymidine (dThd) forming thymidine monophosphate (dTMP). L. major Type II TK (LmTK) has been previously shown to be important for i...
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ftdoajarticles:oai:doaj.org/article:3756b357c9194de8b705af699ce4ab89 2023-05-15T15:12:25+02:00 Structural and Kinetic Characterization of Thymidine Kinase from Leishmania major. Jennifer Timm Cristina Bosch-Navarrete Eliseo Recio Joanne E Nettleship Heather Rada Dolores González-Pacanowska Keith S Wilson 2015-05-01T00:00:00Z https://doi.org/10.1371/journal.pntd.0003781 https://doaj.org/article/3756b357c9194de8b705af699ce4ab89 EN eng Public Library of Science (PLoS) http://europepmc.org/articles/PMC4433323?pdf=render https://doaj.org/toc/1935-2727 https://doaj.org/toc/1935-2735 1935-2727 1935-2735 doi:10.1371/journal.pntd.0003781 https://doaj.org/article/3756b357c9194de8b705af699ce4ab89 PLoS Neglected Tropical Diseases, Vol 9, Iss 5, p e0003781 (2015) Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 article 2015 ftdoajarticles https://doi.org/10.1371/journal.pntd.0003781 2023-01-08T01:27:25Z Leishmania spp. is a protozoan parasite and the causative agent of leishmaniasis. Thymidine kinase (TK) catalyses the transfer of the γ-phosphate of ATP to 2'-deoxythymidine (dThd) forming thymidine monophosphate (dTMP). L. major Type II TK (LmTK) has been previously shown to be important for infectivity of the parasite and therefore has potential as a drug target for anti-leishmanial therapy. In this study, we determined the enzymatic properties and the 3D structures of holo forms of the enzyme. LmTK efficiently phosphorylates dThd and dUrd and has high structural homology to TKs from other species. However, it significantly differs in its kinetic properties from Trypanosoma brucei TK since purines are not substrates of the enzyme and dNTPs such as dUTP inhibit LmTK. The enzyme had Km and kcat values for dThd of 1.1 μM and 2.62 s(-1) and exhibits cooperative binding for ATP. Additionally, we show that the anti-retroviral prodrug zidovudine (3-azido-3-deoxythymidine, AZT) and 5'-modified dUrd can be readily phosphorylated by LmTK. The production of recombinant enzyme at a level suitable for structural studies was achieved by the construction of C-terminal truncated versions of the enzyme and the use of a baculoviral expression system. The structures of the catalytic core of LmTK in complex with dThd, the negative feedback regulator dTTP and the bi-substrate analogue AP5dT, were determined to 2.74, 3.00 and 2.40 Å, respectively, and provide the structural basis for exclusion of purines and dNTP inhibition. The results will aid the process of rational drug design with LmTK as a potential target for anti-leishmanial drugs. Article in Journal/Newspaper Arctic Directory of Open Access Journals: DOAJ Articles Arctic Holo ENVELOPE(9.954,9.954,63.343,63.343) PLOS Neglected Tropical Diseases 9 5 e0003781 |
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ftdoajarticles |
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English |
topic |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
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Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 Jennifer Timm Cristina Bosch-Navarrete Eliseo Recio Joanne E Nettleship Heather Rada Dolores González-Pacanowska Keith S Wilson Structural and Kinetic Characterization of Thymidine Kinase from Leishmania major. |
topic_facet |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
description |
Leishmania spp. is a protozoan parasite and the causative agent of leishmaniasis. Thymidine kinase (TK) catalyses the transfer of the γ-phosphate of ATP to 2'-deoxythymidine (dThd) forming thymidine monophosphate (dTMP). L. major Type II TK (LmTK) has been previously shown to be important for infectivity of the parasite and therefore has potential as a drug target for anti-leishmanial therapy. In this study, we determined the enzymatic properties and the 3D structures of holo forms of the enzyme. LmTK efficiently phosphorylates dThd and dUrd and has high structural homology to TKs from other species. However, it significantly differs in its kinetic properties from Trypanosoma brucei TK since purines are not substrates of the enzyme and dNTPs such as dUTP inhibit LmTK. The enzyme had Km and kcat values for dThd of 1.1 μM and 2.62 s(-1) and exhibits cooperative binding for ATP. Additionally, we show that the anti-retroviral prodrug zidovudine (3-azido-3-deoxythymidine, AZT) and 5'-modified dUrd can be readily phosphorylated by LmTK. The production of recombinant enzyme at a level suitable for structural studies was achieved by the construction of C-terminal truncated versions of the enzyme and the use of a baculoviral expression system. The structures of the catalytic core of LmTK in complex with dThd, the negative feedback regulator dTTP and the bi-substrate analogue AP5dT, were determined to 2.74, 3.00 and 2.40 Å, respectively, and provide the structural basis for exclusion of purines and dNTP inhibition. The results will aid the process of rational drug design with LmTK as a potential target for anti-leishmanial drugs. |
format |
Article in Journal/Newspaper |
author |
Jennifer Timm Cristina Bosch-Navarrete Eliseo Recio Joanne E Nettleship Heather Rada Dolores González-Pacanowska Keith S Wilson |
author_facet |
Jennifer Timm Cristina Bosch-Navarrete Eliseo Recio Joanne E Nettleship Heather Rada Dolores González-Pacanowska Keith S Wilson |
author_sort |
Jennifer Timm |
title |
Structural and Kinetic Characterization of Thymidine Kinase from Leishmania major. |
title_short |
Structural and Kinetic Characterization of Thymidine Kinase from Leishmania major. |
title_full |
Structural and Kinetic Characterization of Thymidine Kinase from Leishmania major. |
title_fullStr |
Structural and Kinetic Characterization of Thymidine Kinase from Leishmania major. |
title_full_unstemmed |
Structural and Kinetic Characterization of Thymidine Kinase from Leishmania major. |
title_sort |
structural and kinetic characterization of thymidine kinase from leishmania major. |
publisher |
Public Library of Science (PLoS) |
publishDate |
2015 |
url |
https://doi.org/10.1371/journal.pntd.0003781 https://doaj.org/article/3756b357c9194de8b705af699ce4ab89 |
long_lat |
ENVELOPE(9.954,9.954,63.343,63.343) |
geographic |
Arctic Holo |
geographic_facet |
Arctic Holo |
genre |
Arctic |
genre_facet |
Arctic |
op_source |
PLoS Neglected Tropical Diseases, Vol 9, Iss 5, p e0003781 (2015) |
op_relation |
http://europepmc.org/articles/PMC4433323?pdf=render https://doaj.org/toc/1935-2727 https://doaj.org/toc/1935-2735 1935-2727 1935-2735 doi:10.1371/journal.pntd.0003781 https://doaj.org/article/3756b357c9194de8b705af699ce4ab89 |
op_doi |
https://doi.org/10.1371/journal.pntd.0003781 |
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PLOS Neglected Tropical Diseases |
container_volume |
9 |
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5 |
container_start_page |
e0003781 |
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