Engineering kinases for dual thymidine and thymidylate kinase activity Público
Liu, Yichen (2011)
Abstract
Engineering kinases for dual thymidine and thymidylate kinase
activity
By Yichen Liu
Thymidine kinase (TK) and thymidylate kinase (TMPK) are essential
enzymes
that function in the salvage pathway for synthesis of thymidine
triphosphate, a DNA
building block. Their importance also exists in activating
nucleoside analogs (NAs), an
important category of anti-cancer and anti-virus drugs. Because NAs
enter the cell as
prodrugs, they require three consecutive phosphorylation steps
catalyzed by cellular
kinases in the salvage pathway to become biologically active and
halt DNA synthesis.
Among the cellular kinases responsible for NA activation, the first
two
phosphorylation steps usually present the bottleneck because of
their poor activity
towards NA. Previous engineering efforts to alter and improve
specificity of these
kinases have largely focused on the first step of phosphorylation
catalyzed by
deoxyribonucleoside kinase (dNK). However, such approach bears the
real risk of simply
shifting the bottleneck from the improved first to the second
phosphorylation reaction. A
more promising strategy is to eliminate both bottleneck steps by
creating a dual
functioning enzyme that can convert NAs directly to their
corresponding diphosphate
form. In this dissertation, I developed a conditional auxotroph
E. coli strain for the
selection of dual TK and TMPK activity. As the first selection
system reported for
selecting two consecutive reactions, this auxotroph strain enables
the evaluation of
combinatorial kinase libraries for TK and TMPK activities both
independently and
concurrently. Employing the dual-function auxotroph strain, I have
evaluated chimera
libraries of dNK from Drosophila melanogaster and TMPK from
Thermotoga maritima
using the non-homologous recombination technique SCRATCHY in
combination with
the computational method SCHEMA. Separately, more fundamental
questions
concerning the mechanistic similarities and differences between
these two enzymatic
reactions were investigated by site-directed mutagenesis and ITC
studies of substrate
binding. Experiments were conducted on DmdNK and TmTMPK, which
excusively
catalyze the first or second phosphorylation reaction, as well as
the only know dual-
function TK from herpes simplex virus (HSV). The information
extracted from these
experiments provides new insights regarding the dual function of
HSV1-TK and will
guide future engineering experiments.
Table of Contents
Table of Contents
Chapter 1 General introduction.
.............................................................................
1
1.1 Nucleoside analog as anti-cancer and anti-virus treatment.
.................................. 2
1.2 Problems in NA activation by cellular salvage pathway and gene
therapy .......... 5
1.3 Deoxyribonucleoside kinase and nucleoside monophosphate kinase
................... 9
1.3.1 dNK family
...................................................................................................
9
1.3.2 NMPK family
.............................................................................................
14
1.3.3 Multifunctional enzymes
............................................................................
19
1.4 Engineering multifunctional kinase and the limitation
....................................... 20
1.5 Aim and scope of the dissertation
.......................................................................
23
Chapter 2 Expression and characterization of thymidylate kinase
from
Thermotoga maritima
................................................................................................
24
2.1 Introduction.
.........................................................................................................
25
2.2 Results and discussion
..........................................................................................
30
2.2.1 Structure and active site of TmTMPK
........................................................
30
2.2.2 TmTMPK gene isolation and protein purification
..................................... 34
2.2.3 TmTMPK is a highly thermostable thymidylate kinase
............................. 36
2.2.4 TmTMPK in vitro catalytic performance
................................................... 38
2.2.5 Isothermal Titration Calorimetry (ITC) of TmTMPK
................................ 40
2.3 Conclusion remarks
.............................................................................................
45
2.4 Materials and methods
........................................................................................
46
2.4.1 Materials
.....................................................................................................
46
2.4.2 Isolation of TmTMPK gene
........................................................................
46
About this Dissertation
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