Chemical probes for mechanistic enzymology

Scientists have been fascinated by enzymatic catalysis for centuries and still marvel at the efficiency and selectivity with which these highly evolved molecular machines are able to catalyze complex reactions. For chemists, understanding the precise catalytic mechanisms can open up new possibilities in synthetic chemistry. From a medicinal standpoint, mechanistic knowledge provides valuable information on how enzymes can be targeted by drugs, as the molecular basis for many diseases lies in enzymatic malfunction. Among the different techniques used by researchers to investigate enzymatic catalysis, chemical probes are an indispensable tool. By synthetically inserting chemical changes into the substrate of a target enzyme, a wealth of information can be gained on structural and mechanistic features. In this thesis we investigate three enzymes with the help of chemical probes. The first and second chapters focus on the first two steps in the biosynthetic pathway of ergothioneine, an essential thiohistidine with antioxidant properties. The third chapter focuses on a key enzyme in the maturation of sulfatases, inserting a unique and crucial catalytic residue into the latter’s active site. In the first chapter, we shed light on the molecular toolbox of the SAM-dependent methyltransferase EgtD. This enzyme catalyzes the first step in the synthesis of ergothioneine, the processive methylation of histidine to Nα,Nα,Nα-trimethyl histidine. By labelling the substrates with stable isotopes, we have created probes enabling us to decipher the molecular strategies which allow this enzyme to carry out three consecutive methylation steps with nearly equal efficiency. In the second chapter we investigate the sulfoxide synthase EgtB, a non-heme iron dependent enzyme catalyzing the second step in ergothioneine biosynthesis. Interestingly, this system can potentially catalyze two distinct reactions, namely C-S bond formation and thiol dioxygenation. The factors governing this bifurcation in reactivity were investigated using substrate analogs with inhibitory properties. In the third and final chapter we examine the formylglycine generating enzyme (FGE), a copper-dependent enzyme catalyzing oxidative C-H bond cleavage of a cysteine residue in the active site of sulfatases. By synthesizing substrate peptides carrying stereoselective deuterium-labels we have identified the rate limiting step of this reaction and have gained valuable insight into the geometry of the enzyme active site

Characterization of competition between commensal and clinical strains of Enterococcus faecium

Invasive infection by multidrug-resistant Enterococcus faecium is increasingly becoming a healthcare concern. Few studies have addressed whether and how clinical E. faecium strains are able to outcompete commensal strains. This thesis aimed to characterize the in vitro competition between commensal and clinical E. faecium strains with respect to growth inhibition exerted by bacteriocins produced by the respective strain groups. A combination of laboratory assays and bioinformatical methods were applied toward this goal. Laboratory assays consisted of competitively growing commensal and clinical E. faecium strains, and using unmodified and modified supernatants from the same strains to investigate the nature of growth inhibition-mediating agents. The bioinformatical methods consisted of genetic relationship analyses and ribosomally synthesized post-translationally modified peptide-mining. It was shown that clinical E. faecium strains generally outcompete commensal E. faecium strains in vitro, though the commensal E. faecium strains that inhibit the growth of clinical E. faecium strains most often, do so very strongly. Furthermore, some commensal and clinical E. faecium strains possibly encoding novel bacteriocins were identified. Finally some commensal E. faecium strains that were resistant to the growth-inhibiting mechanisms of most epidemic and hospital-associated clinical E. faecium strains were found

Grundlagen des Verwaltungsrechts : Forschungsfragen und Forschungskontexte

Im Bereich der Grundlagen des Verwaltungsrechts stellen sich zahlreiche Forschungsfragen. Sie betreffen beispielsweise die Steuerung der Verwaltungstätigkeit, die Methodik des Verwaltungshandelns oder die Geschichte des Verwaltungsrechts. Die Erforschung dieser Themen wäre besonders deshalb wichtig, weil die Verwaltungsrechtswissenschaft weiterhin vom Bild einer einseitig-deduktiven Rechtsanwendung geprägt ist, das die Realität zu sehr vereinfacht. Die Forschung in diesen Bereichen gestaltet sich als schwierig, vor allem weil sie zumindest teilweise empirisch verfahren müsste. Eine entsprechende Forschungsmethodik ist in der Verwaltungsrechtswissenschaft und in der Rechtswissenschaft generell kaum etabliert. Der Ausweg einer verstärkten interdisziplinären Zusammenarbeit stösst auf praktische Schwierigkeiten. Zudem fehlt ein stabiler institutioneller Ort der verwaltungsrechtswissenschaftlichen Grundlagenforschung. Daher dürfte die Forschung in diesem Bereich auch in Zukunft eher punktuellen Charakter haben.Many research questions arise with regard to the basic principles of administrative law. They touch upon, for instance, control and direction of administrative action, its methodology or the history of administrative law. Research into these issues would be especially important because administrative jurisprudence is still dominated by the image of a one-sided deductive application of the law, an image that is overly simplistic. But it is difficult to design research into these areas, above all because they would have to, at least partially, proceed empirically. The research methodology that such empiric study would call for, however, is barely established within administrative law studies and even legal studies more generally. The way out of this impasse offered by interdisciplinary cooperation itself encounters practical difficulties. Furthermore, administrative law research lacks a stable institutional home. Future research in this area thus will probably be tend to be piecemeal at best

Politische und rechtliche Gründe : Notizen zur Pluralität des Begründens

Im philosophischen Diskurs der Gegenwart spielt das Thema der Gründe und der Begründungen eine zentrale Rolle. Vernünftiges Handeln zeichnet sich nach vielfach vertretener Auffassung dadurch aus, dass es dafür gute Gründe gibt, und vernünftiges Interagieren ist durch Prozesse des Gründe‐Gebens und Gründe‐Akzeptierens bestimmt. Im Beitrag soll betrachtet werden, welche Gründe eine politische und welche eine juristische Argumentation stützen und wie diese Formen des Begründens sich zueinander verhalten

Do All Roads Really Lead to Rome? Learnings from Comparative Analysis using SPR, NMR, & X-Ray Crystallography to Optimize Fragment Screening in Drug Discovery.

There are several biophysical methods developed to rapidly identify weakly binding fragments to a target protein. X-ray crystallography provides structural information that is crucial for fragment optimization, however there are several criteria that must be met for a successful fragment screening including the production of soakable and well-diffracting crystals. Therefore, having a reliable cascade of screening methods to be used as pre-screens prior to labor-intensive X-ray crystallography would be extremely beneficial. This would allow the filtering of compounds as the screening progresses so that only the most promising hits remain. But which method should be the one to start the screening cascade? In this work, various sets of fragment libraries were screened against three different proteins; namely tRNA guanine transglycosylase (TGT) an important protein in Shigella, membrane associated protein peroxin 14 (PEX14) of T. Brucei, and endothiapepsin (EP), to investigate whether different screening methods will reveal similar collections of putative binders. The detailed comparative analysis of the findings obtained by the different methods is discussed in this thesis. Shigellosis, an acute bacterial infection of the intestine, is caused by the gram-negative Shigella bacterium whose pathogenicity is reliant on virulence factors (VirF) required to invade epithelial cells. The expression of these VirF is modulated by TGT. Strategies developed to inhibit TGT include potent active-site inhibitors to block the binding of tRNA, thereby preventing the transcription of the virulence factors. Our 96-fragment library was screened against TGT using SPR, NMR, and X-ray crystallography, as described in Chapter 2. A total of 81 fragments were screened in SPR using a direct binding assay approach, revealing a hit rate of 12%. A total of 77 fragments were screened in NMR revealing a hit rate of 29%. High-resolution crystal structures were also collected for the entire fragment library by soaking, revealing a hit rate of 8%. Upon comparison of all discovered fragment hits no overlaps from all three methods were found. Several factors are responsible for this finding such as exclusion of fragments from individual screens due to technical reasons. In detail, four X-ray hits were excluded from the SPR and NMR screens, two SPR hits were discarded from the NMR screen, and five NMR hits were never subjected to the SPR screen. SPR and NMR are currently the most commonly applied primary fragment screening techniques, however, our results suggest that if they would have been applied as incipient methods of a screening cascade, they would have missed three binders discovered by a subsequently applied, more elaborate crystallographic screen. X-ray crystallography allows the detection of specific binders that may be too weak binders to be detected by SPR and even by NMR but can still provide valid structural information to support the search for appropriate starting points in lead discovery. Additionally, MD simulations of the apo wild type TGT have predicted the opening of a transient sub-pocket located above the guanine/preQ1 pocket, which suggested a strategy to target this new binding site for the design of new inhibitors against TGT following a structure- based drug design concept which is also discussed in section 2.3. The human African trypanosomiasis (HAT), also known as the sleeping sickness, is a vector-borne parasitic disease caused by T. brucei and transmitted to humans by bites of the tsetse fly. T. brucei lacks feedback allosteric regulation of early steps in glycolysis but compartmentalizes the relevant enzymes within organelles called glycosomes. PEX14, a peroxin protein essential for biogenesis of glycosomes, forms an important protein-protein interaction with PEX5, an import receptor that transports cytoplasmic glycosomal enzymes into the organelle. Disrupting the PEX14/PEX5 interaction leads to the accumulation of glycosomal enzymes in the cytosol, depletion of ATP, glucose toxicity, metabolic collapse and death of T. brucei. This disruption can be achieved through small molecules that bind to and block PEX14, preventing PEX5 binding. A previous NMR screening of a fragment library resulted in fragment hits that bind to the N-terminal domain (NTD) of T. brucei PEX14. In this project, we attempted to validate these hits through X-ray crystallography by soaking, to allow visualization of the fragment interactions. The promising fragment hits would then be optimized into more potent lead compounds. Crystallization of the NTD PEX14 with a mutation in the first residue (E1W) revealed blocked binding pockets, as described in Chapter 3. The purpose of the added tryptophan was to render fluorescent properties to the short NTD construct which lacked fluorescent amino acids. However, this tryptophan was found to block the binding pockets of its neighboring crystal mates in the protein crystal, rendering a crystal form impossible to use for soaking. Attempts to find new crystal forms with free pockets were unsuccessful, as the small size of the protein and the hydrophobic nature of tryptophan rendered tightly packed protein crystals that block the binding pockets of neighboring crystal mates. Virtual Screening to discover novel ligands for co-crystallization revealed a ligand that aids the crystallization of the E1W PEX14 variant in the same space group but with a slightly different packing. This produced a crystal form that proved successful for fragment soaking as it enabled the binding of two additional fragment hits binding to further protein pockets. Additionally, the wild type form of PEX14 which lacks the tryptophan residue and thus has free binding pockets was crystallized. This enabled the soaking of a previously designed lead compound in different pockets of the PEX5 binding site. By obtaining a crystal structure of this complex at a resolution of 1.8 Å, the feasibility of using wild type PEX14 crystals for further fragment screening has been demonstrated. Endothiapepsin is a member of the pepsin-like aspartic proteases responsible for the hydrolytic cleavage of peptide substrates. Owing to its high degree of similarity to other pharmacologically relevant aspartic proteases, it has served as the model enzyme for studying their mechanism and to discover first lead structures. In previous work done by other members from our group to identify and characterize endothiapepsin binders, X-ray crystallography was consulted as a primary fragment screening method and its hit identification potential was compared to several biochemical and biophysical screening methods. The fragment library screened was designed for general purposes and contained 361 entries. Comparison of the overlap in the hit rates of the different methods to that of X-ray crystallography revealed a low overlap, with the RDA having the highest overlap at 7% and MS having the lowest overlap at 1% followed by STD NMR at 3%. To understand the reason behind the low overlap, two of these screening techniques were prioritized for closer analysis as described in Chapter 4. The 71 X-ray detected fragment hits were selected and rescreened again with STD NMR under slightly different buffer conditions, in addition to WaterLOGSY NMR experiments. The second STD NMR screen detected almost double the amount of hits as the initial one, and the Water LOGSY screen had the highest correlation from the NMR methods to the X-ray hits at 69%. This comparative analysis also revealed the phenomena of active site fragment displacement by use of so-called reporter ligands and that non-deuterated water in STD NMR may lead to false negatives. The entire 361 fragment library was also screened with SPR using an inhibition in solution assay, adding another biophysical method for our comparative analysis to give us further insight of which conditions are crucial to maintain while transferring across different techniques. The resulting hit rate from SPR was 34%, correlating to an overlap of 11% with the X-ray hits - the highest correlation between screening methods reported by us thus far. Finally, we also studied fragment detection and cocktailing in crystallography in comparison to fragment cocktailing in NMR. From this we concluded that cocktailing in crystallography can also lead to false negatives due to fragment competitive behavior and can reveal a different binding mode for a given fragment compared to the adopted geometry found when soaked individually. As for NMR, despite the ability to detect competitive binding of fragments due to the temporary binding and unbinding events, the parallel binding and thus detection of fragments is not always guaranteed as seen in 20% of the fragments we screened, in addition to our observation that the detection of fragments in cocktail NMR may also depend on the comparison of the cocktail set they are a part of

Internal geometry of the central Sesia Zone (Aosta Valley, Italy): HP tectonic assembly of continental slices

Detailed field mapping reveals that the Sesia Zone is subdivided into two complexes with the Barmet Shear Zone (BSZ) outlining the tectonic contact between them. This greenschist-facies contact reflects a metamorphic gap between the Internal Complex (eclogite facies, eclogitic micaschists dominant) and the External Complex (epidote blueschist facies). The BSZ comprises a wedge shape area in which fragments and slices of orthogneiss and paragneiss are wrapped by siliceous dolomite marbles displaying a mylonitic foliation. Conspicuous cornieules and high pressure breccias occur along this contact. We propose that the eclogite facies Internal Complex is subdivided into three basement units, called sheets, delimited by discontinuous metasedimentary trails of probable Mesozoic age. Thin monocyclic bands thus separate kilometre scale polycyclic sheets. The External Complex comprises three epidote blueschist facies sheets of comparable size, which are separated by lenses retaining a pre-Alpine high temperature imprint. These weakly overprinted fragments (parts of the classically termed 2DK zone) are aligned along greenschist facies shear zones that separate the gneissic sheets. The BSZ, with a wedge rich in meta-sediments, chiefly siliceous dolomite marbles, is a key element in which fragmentation and reworking of materials from the internal and external complexes are evident. A carbonate breccia occurs in this shear zone, with clasts displaying a HP foliation randomly oriented in a ductile carbonate matrix. Siliceous dolomite marbles appear to have acted as lubricants to accommodate deformation related to the juxtaposition of the two basement complexes during exhumation. We propose a model of the Sesia Zone, with the BSZ as the thrust responsible for the juxtaposition of eclogite facies rocks of the Internal Complex on top of epidote blueschist facies rocks of the External Complex. The two complexes were already assembled when this shear zone became active. The entire stack was finally rotated (40\u201360\ub0) during the Vanzone Phase

Deeply subducted continental fragments - Part 2: Insight from petrochronology in the central Sesia Zone (western Italian Alps)

Subducted continental terranes commonly comprise an assembly of subunits that reflect the different tectono-metamorphic histories they experienced in the subduction zone. Our challenge is to unravel how, when, and in which part of the subduction zone these subunits were juxtaposed. Petrochronology offers powerful tools to decipher pressure–temperature–time (P–T–t) histories of metamorphic rocks that preserve a record of several stages of transformation. A major issue is that the driving forces for re-equilibration at high pressure are not well understood. For example, continental granulite terrains subducted to mantle depths frequently show only partial and localized eclogitization. The Sesia Zone (NW Italy) is exceptional because it comprises several continental subunits in which eclogitic rocks predominate and high-pressure (HP) assemblages almost completely replaced the Permian granulite protoliths. This field-based study comprises both main complexes of the Sesia terrane, covering some of the recently recognized tectonic subunits involved in its assembly; hence our data constrain the HP tectonics that formed the Sesia Zone. We used a petrochronological approach consisting of petrographic and microstructural analysis linked with thermodynamic modelling and U–Th–Pb age dating to reconstruct the P–T–t trajectories of these tectonic subunits. Our study documents when and under what conditions re-equilibration took place. Results constrain the main stages of mineral growth and deformation, associated with fluid influx that occurred in the subduction channel. In the Internal Complex (IC), pulses of fluid percolated at eclogite facies conditions between 77 and 55 Ma with the HP conditions reaching  ∼  2 GPa and 600–670 °C. By contrast, the External Complex (EC) records a lower pressure peak of  ∼  0.8 GPa for 500 °C at  ∼  63 Ma. The juxtaposition of the two complexes occurred during exhumation, probably at  ∼  0.8 GPa and 350 °C; the timing is constrained between 46 and 38 Ma. Mean vertical exhumation velocities are constrained between 0.9 and 5.1 mm year−1 for the IC, up to its juxtaposition with the EC. Exhumation to the surface occurred before 32 Ma, as constrained by the overlying Biella Volcanic Suite, at a mean vertical velocity between 1.6 and 4 mm year−1. These findings constrain the processes responsible for the assembly and exhumation of HP continental subunits, thus adding to our understanding of how continental terranes behave during subduction

The Challenges and Opportunities of Teaching Systems Engineering for Large Scale Engineering Systemse

In this paper, we discuss a course in large scale systems engineering that was developed as part of a twelve-credit Graduate Certificate in Systems Engineering for part-time and returning students. Our focus was on how to work on big, unmanageable, messy problems. Teaching systems engineering for large-scale engineering systems is challenging for many reasons. One of the main challenges is the breadth of material to be addressed within one or two courses. Other challenges result from how students learn about the transitions from the development of a conceptual framework to systems architecture through engineering design, mathematical analysis and eventual construction and delivery