Cell wall remodeling proteins in Mycobacterium tuberculosis : structure, function and inhibition

Abstract: The complex and peculiar cell wall architecture is vital for the survival of M. tuberculosis in the host and therefore an established target for several currently used drugs. Understanding of the cell wall maintenance and the underlying biochemical mechanisms in this pathogen is expected to aid the development and evaluation of novel anti-TB therapies. Within the scope of this thesis peptidoglycan remodeling enzymes including the essential transpeptidase LdtMt2, an NlpC/P60 hydrolase, RipA and a non-catalytic NlpC/P60 variant, RipD were investigated. LdtMt2, essential in M. tuberculosis for intra-host survival, forms the prevalent 3-3 cross-links within the cell wall peptidoglycan. The structure of LdtMt2 was solved, a model of the three-domain protein located in the periplasm was proposed and the covalent adduct formation with β-lactam antibiotics was observed. The systematic analysis of several β-lactams identified faropenem displaying the fastest binding kinetics and resulting in the formation of a stable adduct. These results and the high-resolution structure of LdtMt2 with this adduct representing the inactivated state describes the detailed action of faropenem, which is the most efficient β-lactam in killing M. tuberculosis in vitro and inside macrophages. During mycobacterial cell division, daughter cell separation requires endopeptidases from the NlpC/P60 protein family. RipD the first non-catalytic member from this family that retains PG binding activity and carries a long penta-peptide repeat sequence in C-terminal position was characterized. RipA comprises a well-characterized C-terminal endopeptidase domain of the NlpC/P60-type and an N-terminal domain of unknown function. The N-terminal domain was previously implicated in inhibition of the catalytic activity by blocking the C-terminal domain. Here we show that it is not the N-terminal domain but the lid-module of the inter-domain linker that limits the active site access. The structure of the N-terminal domain was solved by X-ray crystallography revealing an elongated domain built by two long α-helices. Small angle X-ray scattering in combination with the X-ray structures of the two individual domains was used to model the periplasmic RipA protein suggesting a rigid, hairpin-like N-module connected to the catalytic domain by a flexible linker. This domain organization allows for a defined range of movement of the catalytic domain implicated the spatially controlled cell wall degradation.