Towards the Structural Characterisation of AHK5, an Arabidopsis RedOx sensor and Structural Studies on the Bacterial Cell Wall Modifying Enzyme GatD / MurT

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Dokumentart: Dissertation
Date: 2022-08-29
Language: English
Faculty: 7 Mathematisch-Naturwissenschaftliche Fakultät
Department: Biochemie
Advisor: Stehle, Thilo (Prof. Dr.)
Day of Oral Examination: 2022-02-11
DDC Classifikation: 500 - Natural sciences and mathematics
Keywords: Kristallographie , Strukturbiologie
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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.

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