A fundamental catalytic difference between zinc and manganese dependent enzymes revealed in a bacterial isatin hydrolase

The catalytic mechanism of the cyclic amidohydrolase isatin hydrolase depends on a catalytically active manganese in the substrate-binding pocket. The Mn$^{2+}$ ion is bound by a motif also present in other metal dependent hydrolases like the bacterial kynurenine formamidase. The crystal structures of the isatin hydrolases from Labrenzia aggregata and Ralstonia solanacearum combined with activity assays allow for the identification of key determinants specific for the reaction mechanism. Active site residues central to the hydrolytic mechanism include a novel catalytic triad Asp-His-His supported by structural comparison and hybrid quantum mechanics/classical mechanics simulations. A hydrolytic mechanism for a Mn$^{2+}$ dependent amidohydrolases that disfavour Zn$^{2+}$as the primary catalytically active site metal proposed here is supported by these likely cases of convergent evolution. The work illustrates a fundamental difference in the substrate-binding mode between Mn$^{2+}$ dependent isatin hydrolase like enzymes in comparison with the vast number of Zn$^{2+}$ dependent enzymes

Molecular dynamics study of the mechanical properties of foldamers and the metal specificity of an isatin hydrolase

This thesis presents a theoretical investigation of the mechanical properties of foldamers and the metal specificity of an isatin hydrolase. In the first part, force-probe molecular dynamics simulations are applied to the study of the mechanical unfolding pathway of small oligomers adopting helix conformations. The detailed investigation of a model β-peptide demonstrates how molecular dynamics simulations can be used for revealing conformational and kinetic information of the unfolding pathway of oligomers. The statistical analysis of several hundreds of unfolding events and the comparison between different systems leads to the identification of the main determining structural factors of the unfolding pathway of the studied oligomers. Based on this findings, a series of rules for the prediction of the unfolding pathway of short oligomers are proposed. In the second part, the experimental observed metal specificity of an isatin hydrolase is investigated, using quantum mechanics and quantum- /molecular mechanics calculations. The metal specificity is explained based on the conformation adopted by the enzyme’s binding site with different metal ions and the hydrolysis reaction mechanism. Finally, the mechanism of the catalytic reaction with manganese is revealed using metadynamics simulations.In der vorliegenden Dissertation werden theoretische Untersuchungen zu den mechanischen Eigenschaften von Foldameren sowie zur Metall-Spezifizität eines Enzyms, das die Hydrolyse von Isatin katalysiert, vorgestellt. Im ersten Teil der Arbeit werden Molekulardynamik-Simulationen zur mechanischen Entfaltung der helikalen Konformationen kleinerer Oligomere durchgeführt. Detaillierte Untersuchungen eines β-Peptid-Modellsystems zeigen, wie Molekulardynamik- Simulationen verwendet werden können, um Informationen über Konformationen, Intermediate und kinetische Raten bei der Entfaltung von Foldameren zu erhalten. Eine statistische Analyse mehrerer hundert Entfaltungsereignisse und ein Vergleich verschiedener Modellsysteme erlaubt die Identifizierung der Haupt-Einflussfaktoren auf die Entfaltungspfade der untersuchten Oligomere. Darauf aufbauend wird ein Satz von Regeln vorgeschlagen, mit dessen Hilfe der Entfaltungsmechanismus kurzer Oligomere vorausgesagt werden kann. Im zweiten Teil der Arbeit wird die experimentell beobachtete Metall-Spezifizität einer Isatin-Hydrolase mit Hilfe von quantenmechanischen Rechnungen und quanten-/molekularmechanischen Simulationen untersucht. Die Metall-Spezifizität wird über die unterschiedlichen Konformationen des aktiven Zentrums des Enzyms mit den verschiedenen Metallionen sowie über die Anforderungen des Hydrolyse-Reaktionsmechanismus erklärt. Darüberhinaus wird mit Hilfe von Metadynamik-Simulationen der Hydrolyse-Mechanismus entschlüsselt

Fast and shift-insensitive similarity comparisons of NMR using a tree-representation of spectra

An efficient method to extract and store information from NMR spectra is proposed that is suitable for comparison and construction of a search engine. This method based on trees doesn't require any peak picking or any pre-treatment of the data and is found to outperform the currently available methods, both in terms of compactness and velocity. Our approach was tested for 1D proton spectra and 2D HSQC spectra and compared with the method proposed by Pretsch and coworkers [1,2] [Bodis et al. 2007, Bodis et al. 2009]. Additionally, the correspondence between spectral and structural similarity was evaluated for both methods. (C) 2013 Elsevier By. All rights reserved

Comparative Study of the Mechanical Unfolding Pathways of α- and β‑Peptides

Using molecular simulations, we analyze the unfolding pathways of various peptides. We compare the mechanical unfolding of a β-alanine’s octamer (β-HAla₈) and an α-alanine’s decamer (α-Ala₁₀). Using force-probe molecular-dynamics simulations, to induce unfolding, we show that the 3₁₄-helix formed by β-HAla₈ is mechanically more stable than the α-helix formed by α-Ala₁₀, although both structures are stabilized by six hydrogen bonds. Additionally, computations of the potential of mean force validate this result and show that also the thermal stability of the 3₁₄-helix is higher. It is demonstrated that β-HAla₈ unfolds in a two-step fashion with a stable intermediate. This is contrasted with the known single-step scenario of the unfolding of α-Ala₁₀. Furthermore, we present a study of the chain-length dependence of the mechanical unfolding pathway of the 3₁₄-helix. The calculation of the dynamic strength for oligomers with chain lengths ranging from 6 to 18 monomers shows that the unfolding pathway of helices with an integer and noninteger number of turns has <i>m</i> + 1 and <i>m</i> energy barriers, respectively, with <i>m</i> being the number of complete turns. The additional barrier for helices with an integer number of turns is shown to be related to the breaking of the N-terminus’ hydrogen bond

Determining Factors for the Unfolding Pathway of Peptides, Peptoids, and Peptidic Foldamers

We present a study of the mechanical unfolding pathway of five different oligomers (α-peptide, β-peptide, δ-aromatic-peptides, α/γ-peptides, and β-peptoids), adopting stable helix conformations. Using force-probe molecular dynamics, we identify the determining structural factors for the unfolding pathways and reveal the interplay between the hydrogen bond strength and the backbone rigidity in the stabilization of their helix conformations. On the basis of their behavior, we classify the oligomers in four groups and deduce a set of rules for the prediction of the unfolding pathways of small foldamers