Abstract:
This thesis presents recent advances on the structural and mechanistical characterisation of
two ATP-binding proteins or protein complexes; AHK5 from Arabidopsis thaliana, and GatD/MurT from
Staphylococcus aureus, Mycobacterium tuberculosis, and Streptococcus pyogenes.
AHK5 is a sensory histidine kinase that is involved in multiple physiological pathways, such as
the modulation of stomatal closure, or the response to PAMPs. Its activity is likely triggered by increases
in hydrogen peroxide concentrations. In this project, a tentative model for hydrogen peroxide
sensing by the N-terminal portion of AHK5 was postulated. Based on in-vitro dimerisation studies, a
cysteine residue at position 3 was shown to covalently link two AHK5 monomers under oxidative conditions,
with a redox midpoint potential compatible with previously published physiological values.
The study additionally lays a comprehensive foundation for future work to elucidate the structure and
thus the details of AHK5 activation. Three different fragments which, together, span the sensory Nterminus
and the histidine kinase and ATPase domains were successfully purified. Bioinformatic methods
as well as a protease exclusion assay indicated the probable presence of a yet undescribed PASlike
domain, immediately preceding the histidine kinase domain. This domain could play a key role in
transducing the input signal from the N-terminus to the catalytically active part. However, biophysical
analyses highlight potential heterogeneity with respect to the purified proteins’ oligomeric state and
stability problems. These issues will be the subject of follow-up studies before structural work can
begin.
Understanding the mechanism of action of AHK5 will not only provide additional understanding
of how histidine kinases work; due to its key role in numerous physiological processes, it could
prove an anchor point from which to deepen our understanding of how Arabidopsis reacts to exogenous
stresses as well as plant physiology in general.
GatD/MurT is a cell wall amidating enzyme complex that is essential in many Gram-positive
bacteria, including highly pathogenic ones like Staphylococcus aureus, Streptococcus pneumoniae, and
even the actinobacterium Mycobacterium tuberculosis. In this project, the first fully refined crystal
structure of the S. aureus enzyme was obtained and a complex crystal structure containing an ATP
analogue mimicking one of the three substrates was solved. Together with solution scattering data
obtained by SAXS, these data indicate an intriguing crescent-shaped open conformation of the heterodimer.
This conformation likely represents the enzyme’s resting state and probably transitions to a
more compact conformation upon substrate binding, in order to achieve catalysis. The enzyme complex
exhibits a canonical ATP binding pocket in MurT. GatD achieves intermediate ammonia generation
by deamidation of cytosolic glutamine. This reaction is catalysed by an unusual intermolecular catalytic
triad composed of a cysteine and a histidine in GatD and an aspartate in MurT. Additionally, MurT
contains a Cys4-type Zinc finger of unknown function. Expression of putative homologous complexes
from M. tuberculosis and Streptococcus pyogenes was successful, and preliminary glutaminase activity
monitored by 1H-NMR confirmed both enzymes to be active in this respect.
These insights raise the stakes in understanding the molecular mechanism of action of
GatD/MurT, as it could offer the possibility to develop novel antimicrobial drugs that specifically target
it. Such drugs could be active not only against S. aureus but rather a wide range of pathogenic bacteria,
thus providing a new edge in combating antibiotic resistances.
Print-on-Demand