NMR studies of Mycobacterium Tuberculosis Adenylate kinase mutants
with ATP and AMP titration
SeongWon Han, HyunJu Choi, SeonHui Woo, HoSik Won
Department of applied chemistry Hanyang University
Adenylate kinase from Mycobacterium tuberculosis (AKmt), a protein of 181
residues is short variant with low similarity with the eukaryote cytosolic AKs. It is an
essential enzyme for bacterial survival and it’s catalytic activity is lower than other
AKs((AKe, AK1 etc). But recently we found that catalytic activity of two point double
mutant(A11S, E122K) of AKmt, AKmtDM was 12 fold increased. Adenylate
kinases(AKs) enzymes which generate ADP through catalyzing reversible high
energy phosphorylation between ATP and AMP (ATP•Mg2+ + AMP ⇔ ADP•Mg2+ +
ADP )are known to be associated with energy metabolism and nucleic acid synthesis.
So, Point of our study is investigating structure and binding characteristic of
AkmtDM . We can obtain AkmtDM protein through genetic recombination of Akmt,
and do cell culture was carried out to produce a 15N-labeled mutant protein. In this
work a solution-state NMR approach and Molecular Dynamics simulation were used
to probe the structural and dynamic distinction of AKmtDM. Firstly, chemical shift
analysis of AKmtDM, using the 1H-15N HSQC experiment, show that ATP binding Ploop(7-13) and LID domain and include LID domain surrounding residues(115~135),
are slightly perturbed(δw≈0.1~03ppm). It suggests that AKmtDM backbone structure
is differ from AKmt at ATP binding active site and it’s structural perturbation maybe
induce significant backbone rearrangement and eventually produce the difference of
catalytic activity. MD simulation also show that stable salt bridge K122-E118 at the
LID domain interface and hydrogen bonding R123-S11 LID-Core interaction stabilize
the open conformation.
These results provide specific knowledge about structural dynamic motion of
AKmt/AKmtDM and more generally suggest that molecular dynamics simulation can
provide valuable information for identifying catalytic activity.
Structural and Dynamic studies on Ligand bound single Mutant
Adenylate Kinase from Mycobacterium Tuberculosis
Asghar Khan, HoSik Won
Department of applied chemistry Hanyang University
Adenylate Kinase of mycobacterium tuberculosis (Akmt) is a ubiquitous
enzyme, a protein of 181 residues that catalyses a reversible high energy phosphoryl
transfer reaction between ATP and AMP to form ADP.
In the present study, single mutant 15N labeled Akmt gene was cloned,
express in E.coli and purified as Akmt-11S.The Akmt-11S was studied with 1H and
15N NMR relaxation experiments, aiming to characterize the solution structure,
binding mechanism and internal dynamics of the various domains. Analysis of the
15N relaxation data enabled us to obtain the relaxation rate constants R1, R2 and
global rotational correlation time (τm), which explains the dynamic motion and
conformational change of the Akmt-11S enzyme on binding with AMP and ATP
ligands.
In support with NMR data at 10ns MD simulation was also run to closely
explain the dynamic motion, calculate RMSD, RMSF and principal component
analysis. In the last the results of single mutant Akmt-11S was compared with wild
type Akmt.