Advanced

The Structure-Function Relationship and Evolution of Deoxyribonucleoside Kinases

Mutahir, Zeeshan LU (2013)
Abstract (Swedish)
Popular Abstract in English

Deoxyribonucleoside triphosphates (dNTPs) are the building blocks of DNA and the balanced cellular dNTP pools are of extreme importance to maintain the genomic stability and overall function of the cell. Primarily, two different pathways exist in most of the organisms that are responsible for providing dNTPs. One is called the de novo pathway where dNTPs are formed from scratch in a series of biochemical reactions using basic molecules like sugars, amino acids, CO2, etc. The other mean of providing dNTPs is known as salvage pathway which is less energy demanding than de novo synthesis. In the salvage pathway, dNTPs are formed by salvaging the intra- and extra-cellular precursors for DNA... (More)
Popular Abstract in English

Deoxyribonucleoside triphosphates (dNTPs) are the building blocks of DNA and the balanced cellular dNTP pools are of extreme importance to maintain the genomic stability and overall function of the cell. Primarily, two different pathways exist in most of the organisms that are responsible for providing dNTPs. One is called the de novo pathway where dNTPs are formed from scratch in a series of biochemical reactions using basic molecules like sugars, amino acids, CO2, etc. The other mean of providing dNTPs is known as salvage pathway which is less energy demanding than de novo synthesis. In the salvage pathway, dNTPs are formed by salvaging the intra- and extra-cellular precursors for DNA synthesis. Deoxyribonucleoside kinases (dNKs) are the enzymes which catalyze the first and rate determining step of the salvage pathway and play an important role in maintaining the balanced dNTP pools. There are four different dNKs in humans: thymidine kinase 1 (TK1), thymidine kinase 2 (TK2), deoxycytidine kinase (dCK) and deoxyguanosine kinase (dGK). Based on their three dimensional structures and amino acid sequence similarities, the dNKs can broadly be divided into two families: the TK1-like and the non-TK-1/TK2-like families. The dNKs show distinct but overlapping substrate specificities. The TK1-like enzymes have strict substrate specificity compared to the non-TK1-like enzymes which show broad substrate specificity. The dNKs are of immense medical importance because they can activate nucleoside analogues, used as anti-viral and anti-cancer drugs, into toxic compounds. In addition, dNKs present an excellent model to study different evolutionary phenomena, such as, the origin of regulatory activities, gene duplications and losses. Keeping in view the importance of dNKs, the present research aimed at furthering the understanding of diversity, structure-function relationship and evolutionary background of these enzymes. Numerous molecular biology, biochemistry and bioinformatics’ tools were employed to characterize different dNKs, mainly from vertebrates and plants. The results obtained here provide the basis for future research possibilities.

Human TK1 is highly regulated enzyme and has been reported to exist in two forms: a dimer and a tetramer form. The two forms show different catalytic efficiency for its natural substrate i.e. thymidine. The tetramer form is 30-fold more efficient than dimer form and the dimer/tetramer shift is dependent on enzyme concentration and ATP. Furthermore, the oligomerization shift of human TK1 is considered as one of the regulatory mechanisms of the enzyme. So far, the oligomerization shift has been described in a couple of TK1s. In this study, the ‘ATP-induced’ oligomerization switch on TK1s from distantly related organisms was analyzed to understand structure-function relationship and the evolutionary origin of oligomerization based regulation of TK1. The results suggested that the dimer form is the ancestral form and the tetramer form has originated as an additional regulatory control in animals.

In addition, the phylogenetic distribution and evolutionary history of non-TK1-like dNKs among vertebrates was studied. A duplicated dCK homolog was found in different vertebrate lineages and loss of dGK has been observed in one of the bird families. In order to explore the possible biological forces behind gene duplication of dCK and loss of dGK, kinetic properties and substrate specificities of recombinant dCK/dGK-like enzymes from an amphibian, Xenopus laevis, and a bird, Gallus gallus, were determined. Moreover, considering the need of balanced dNTP pools, both in cytosol and mitochondria, sub-cellular localization of the abovementioned enzymes was predicted. The results proposed that substrate specificity and the sub-cellular localization of these enzymes are the likely forces behind vertebrate dCK/dGK evolution. (Less)
Abstract
The synthesis of deoxyribonucleoside triphosphates (dNTPs), building blocks of DNA, can be achieved by two different pathways in the cell. One is called de novo synthesis and the other is known as salvage pathway. The de novo pathway involves synthesis of dNTPs by utilizing basic molecules like sugars, amino acids, CO2, NH3, etc., while in the other pathway, dNTPs are formed by salvaging intra- or extra-cellular deoxyribonucleosides (dNs). Deoxyribonucleoside kinases (dNKs) catalyze the rate-limiting step of the nucleoside salvage pathway by converting the dNs into the corresponding deoxyribonucleoside monophosphates and play an important role in maintaining the balanced dNTP pools in the cell. Furthermore, dNKs show a great deal of... (More)
The synthesis of deoxyribonucleoside triphosphates (dNTPs), building blocks of DNA, can be achieved by two different pathways in the cell. One is called de novo synthesis and the other is known as salvage pathway. The de novo pathway involves synthesis of dNTPs by utilizing basic molecules like sugars, amino acids, CO2, NH3, etc., while in the other pathway, dNTPs are formed by salvaging intra- or extra-cellular deoxyribonucleosides (dNs). Deoxyribonucleoside kinases (dNKs) catalyze the rate-limiting step of the nucleoside salvage pathway by converting the dNs into the corresponding deoxyribonucleoside monophosphates and play an important role in maintaining the balanced dNTP pools in the cell. Furthermore, dNKs show a great deal of multiplicity among different organisms and present an excellent choice to study different evolutionary phenomena, such as, the origin of regulatory activities and gene/enzyme duplication.

The present study deals with the characterization of different dNKs in order to understand their structure-function relationship, substrate specificities and evolutionary history. The ‘ATP-induced’ dimer/tetramer shift on thymidine kinase 1 (TK1) from different organisms was studied to comprehend the evolutionary origin of oligomerization based regulation of TK1. The results suggested that the dimer form is the original form and the tetramer form has originated in early animals to serve as a fine-tuning mechanism for the regulation of TK1 activity.

In addition, the evolutionary background of various dNKs was studied. An apparent duplication of deoxycytidine kinase (dCK) in some vertebrates and loss of deoxyguanosine kinase (dGK) in one of the bird families were observed. The kinetic properties of the recombinant dCK/dGK-like enzymes from an amphibian, Xenopus laevis, and a bird, Gallus gallus, were determined and the sub-cellular localization of these enzymes was predicted. It seems that substrate specificity and sub-cellular localization are the likely biological forces behind vertebrate dCK/dGK evolution.

Finally, the dNK activities in sub-cellular fractions of plants, Arabidopsis thaliana and Solanum tuberosum, were analyzed and the results showed that most of the plant dNK activity mainly occurs in mitochondria, but not in chloroplasts. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • Wang, Liya, Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden
organization
publishing date
type
Thesis
publication status
published
subject
publisher
Department of Biology, Lund University
defense location
Biology building A, Lecture hall, Sölvegatan 35, Lund
defense date
2013-12-19 13:00
ISBN
978-91-7473-730-1
language
English
LU publication?
yes
id
a2e6c09a-0678-4468-ae85-3fdda52a2ced (old id 4157404)
date added to LUP
2013-11-21 08:14:33
date last changed
2016-09-19 08:45:02
@phdthesis{a2e6c09a-0678-4468-ae85-3fdda52a2ced,
  abstract     = {The synthesis of deoxyribonucleoside triphosphates (dNTPs), building blocks of DNA, can be achieved by two different pathways in the cell. One is called de novo synthesis and the other is known as salvage pathway. The de novo pathway involves synthesis of dNTPs by utilizing basic molecules like sugars, amino acids, CO2, NH3, etc., while in the other pathway, dNTPs are formed by salvaging intra- or extra-cellular deoxyribonucleosides (dNs). Deoxyribonucleoside kinases (dNKs) catalyze the rate-limiting step of the nucleoside salvage pathway by converting the dNs into the corresponding deoxyribonucleoside monophosphates and play an important role in maintaining the balanced dNTP pools in the cell. Furthermore, dNKs show a great deal of multiplicity among different organisms and present an excellent choice to study different evolutionary phenomena, such as, the origin of regulatory activities and gene/enzyme duplication.<br/><br>
The present study deals with the characterization of different dNKs in order to understand their structure-function relationship, substrate specificities and evolutionary history. The ‘ATP-induced’ dimer/tetramer shift on thymidine kinase 1 (TK1) from different organisms was studied to comprehend the evolutionary origin of oligomerization based regulation of TK1. The results suggested that the dimer form is the original form and the tetramer form has originated in early animals to serve as a fine-tuning mechanism for the regulation of TK1 activity.<br/><br>
In addition, the evolutionary background of various dNKs was studied. An apparent duplication of deoxycytidine kinase (dCK) in some vertebrates and loss of deoxyguanosine kinase (dGK) in one of the bird families were observed. The kinetic properties of the recombinant dCK/dGK-like enzymes from an amphibian, Xenopus laevis, and a bird, Gallus gallus, were determined and the sub-cellular localization of these enzymes was predicted. It seems that substrate specificity and sub-cellular localization are the likely biological forces behind vertebrate dCK/dGK evolution.<br/><br>
Finally, the dNK activities in sub-cellular fractions of plants, Arabidopsis thaliana and Solanum tuberosum, were analyzed and the results showed that most of the plant dNK activity mainly occurs in mitochondria, but not in chloroplasts.},
  author       = {Mutahir, Zeeshan},
  isbn         = {978-91-7473-730-1},
  language     = {eng},
  publisher    = {Department of Biology, Lund University},
  school       = {Lund University},
  title        = {The Structure-Function Relationship and Evolution of Deoxyribonucleoside Kinases},
  year         = {2013},
}