Peter Kast

Last Name

Kast

First Name

Peter

ORCID

0000-0002-0209-8975

Organisational unit

08816 - Kast, Peter (Tit.-Prof.)

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Publications 1 - 10 of 47

Functional mapping of protein-protein interactions in an enzyme complex by directed evolution
Roderer, Kathrin; Neuenschwander, Martin; Codoni, Giosiana; et al. (2014)
PLoS ONE
The shikimate pathway enzyme chorismate mutase converts chorismate into prephenate, a precursor of Tyr and Phe. The intracellular chorismate mutase (MtCM) of Mycobacterium tuberculosis is poorly active on its own, but becomes >100-fold more efficient upon formation of a complex with the first enzyme of the shikimate pathway, 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (MtDS). The crystal structure of the enzyme complex revealed involvement of C-terminal MtCM residues with the MtDS interface. Here we employed evolutionary strategies to probe the tolerance to substitution of the C-terminal MtCM residues from positions 84–90. Variants with randomized positions were subjected to stringent selection in vivo requiring productive interactions with MtDS for survival. Sequence patterns identified in active library members coincide with residue conservation in natural chorismate mutases of the AroQδ subclass to which MtCM belongs. An Arg-Gly dyad at positions 85 and 86, invariant in AroQδ sequences, was intolerant to mutation, whereas Leu88 and Gly89 exhibited a preference for small and hydrophobic residues in functional MtCM-MtDS complexes. In the absence of MtDS, selection under relaxed conditions identifies positions 84–86 as MtCM integrity determinants, suggesting that the more C-terminal residues function in the activation by MtDS. Several MtCM variants, purified using a novel plasmid-based T7 RNA polymerase gene expression system, showed that a diminished ability to physically interact with MtDS correlates with reduced activatability and feedback regulatory control by Tyr and Phe. Mapping critical protein-protein interaction sites by evolutionary strategies may pinpoint promising targets for drugs that interfere with the activity of protein complexes.
Leveraging relaxation-optimized 1H–13CF correlations in 4-19F-phenylalanine as atomic beacons for probing structure and dynamics of large proteins
Boeszoermenyi, Andras; Radeva, Denitsa L.; Schindler, Sebastian; et al. (2025)
Nature Chemistry
NMR spectroscopy of biomolecules provides atomic level information into their structure, dynamics and interactions with their binding partners. However, signal attenuation from line broadening caused by fast relaxation and signal overlap often limits the application of NMR to large macromolecular systems. Here we leverage the slow relaxation properties of 13C nuclei attached to 19F in aromatic 19F–13C spin pairs as well as the spin–spin coupling between the fluorinated 13C nucleus and the hydrogen atom at the meta-position to record two-dimensional 1H–13CF correlation spectra with transverse relaxation-optimized spectroscopy selection on 13CF. To accomplish this, we synthesized [4-19F13Cζ; 3,5-2H2ε] Phe, engineered for optimal relaxation properties, and adapted a residue-specific route to incorporate this residue globally into proteins and a site-specific 4-19F Phe encoding strategy. This approach resulted in narrow linewidths for proteins ranging from 30 kDa to 180 kDa, enabling interaction studies with small-molecule ligands without requiring specialized 19F-compatible probes.
Preliminary X-ray crystallographic analysis of the secreted chorismate mutase from Mycobacterium tuberculosis
Krengel, Ute; Dey, Raja; Sasso, Severin; et al. (2006)
Acta Crystallographica Section F: Structural Biology and Crystallization Communications
Probing the proteolytic stability of beta peptides containing alpha fluoro- and alpha hydroxy beta amino acids
Hook, David F.; Gessier, François; Noti, Christian; et al. (2004)
ChemBioChem
Structure and function of a complex between chorismate mutase and DAHP synthase
Sasso, Severin; Ökvist, Mats; Roderer, Kathrin; et al. (2009)
The EMBO Journal
Isochorismate Pyruvate Lyase
DeClue, Michael S.; Baldridge, Kim K.; Künzler, Dominik E.; et al. (2005)
Journal of the American Chemical Society
1.6 Å Crystal structure of the secreted chorismate mutase from Mycobacterium tuberculosis
Ökvist, Mats; Dey, Raja; Sasso, Severin; et al. (2006)
Journal of Molecular Biology
Electrostatic transition state stabilization rather than reactant destabilization provides the chemical basis for efficient chorismate mutase catalysis
Burschowsky, Daniel; van Eerde, André; Ökvist, Mats; et al. (2014)
Proceedings of the National Academy of Sciences of the United States of America
For more than half a century, transition state theory has provided a useful framework for understanding the origins of enzyme catalysis. As proposed by Pauling, enzymes accelerate chemical reactions by binding transition states tighter than substrates, thereby lowering the activation energy compared with that of the corresponding uncatalyzed process. This paradigm has been challenged for chorismate mutase (CM), a well-characterized metabolic enzyme that catalyzes the rearrangement of chorismate to prephenate. Calculations have predicted the decisive factor in CM catalysis to be ground state destabilization rather than transition state stabilization. Using X-ray crystallography, we show, in contrast, that a sluggish variant of Bacillus subtilis CM, in which a cationic active-site arginine was replaced by a neutral citrulline, is a poor catalyst even though it effectively preorganizes chorismate for the reaction. A series of high-resolution molecular snapshots of the reaction coordinate, including the apo enzyme, and complexes with substrate, transition state analog and product, demonstrate that an active site, which is only complementary in shape to a reactive substrate conformer, is insufficient for effective catalysis. Instead, as with other enzymes, electrostatic stabilization of the CM transition state appears to be crucial for achieving high reaction rates.
Selective stabilization of the chorismate mutase transition state by a positively charged hydrogen bond donor
Kienhöfer, Alexander; Kast, Peter; Hilvert, Donald (2003)
Journal of the American Chemical Society
Exhaustive Mutagenesis of Six Secondary Active-Site Residues in Escherichia coli Chorismate Mutase Shows the Importance of Hydrophobic Side Chains and a Helix N-Capping Position for Stability and Catalysis
Lassila, Jonathan K.; Keeffe, Jennifer R.; Kast, Peter; et al. (2007)
Biochemistry

Publications 1 - 10 of 47

Peter Kast

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