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Research project (§ 26 & § 27)
Duration : 2018-01-01 - 2020-12-31

Chlorite dismutases (Clds) are able to efficiently decompose chlorite (ClO2-) into harmless chloride (Cl-) and dioxygen (O2) with chlorite being the sole source of dioxygen. Thereby, a covalent oxygen-oxygen bond is formed, a unique biochemical reaction that in addition is only catalyzed by the water-splitting manganese complex of photosystem II of oxygenic phototrophic organisms. The mechanism of cleavage of chlorite is still under heavy debate. Computational studies suggest homolytic cleavage, thereby producing chlorine monoxide (ClO●) and Compound II [Por…Fe(IV)=O] followed by a rebound step and release of chloride and dioxygen, whereas biochemical studies on pentameric (clade 1) Clds suggest heterolytic cleavage of chlorite thereby forming Compound I [Por+●…Fe(IV)=O] and hypochlorite (HOCl/-OCl). However, there is no experimental proof for the generation of Compound I nor is it known why these oxidoreductases have their pH optimum around pH 5 and are inactivated at alkaline pH. In order to elucidate structure-function relationships in Cld and understand the molecular basis of chlorite degradation, we have selected the dimeric Cld from Cyanothece sp. PCC7425 (CCld) as model enzyme for several reasons. CCld can easily be produced in E. coli, allows the generation of crystals of optimum size at all relevant pH values to be probed by both X-ray and neutron crystallography and for the first time showed distinct spectral features of typical heme b Compound II, which was formed immediately after mixing the ferric enzyme with chlorite. Moreover, CCld exhibits further enzymatic features that contradict the proposed heterolytic but favour the homolytic cleavage mechanism of chlorite. Thus it is the aim of this project to fully clarify the molecular mechanism of chlorite cleavage and O2 formation by using a broad set of biochemical and biophysical methods: (i) the recombinant production and purification of the wild-type protein and selected single mutants; (ii) characterization of these iron-proteins by various spectroscopic (resonance Raman, UV-vis) and electrochemical techniques, (iii) analysis of all individual reaction steps of enzyme cycle by multi-mixing stopped-flow spectroscopy and freeze-quench electron paramagnetic resonance (EPR) spectroscopy; and (iv) elucidation of X-ray and neutron crystal structures in the pH range 5-10. The work will be performed in close cooperation with internationally well-known scientists from Austria (X-ray crystallography: Kristina Djinovic-Carugo), Italy (RR spectroscopy: Giulietta Smulevich), Belgium (EPR spectroscopy: Sabine Van Doorslaer) and USA (neutron crystallography: Leighton Coates). Understanding structure-function relationships of Cld will provide the basis for its application in chemical engineering and bioremediation.
Research project (§ 26 & § 27)
Duration : 2017-07-01 - 2021-06-30

Haemonchus contortus is one of the major helminthic parasites, which infects sheep and goats resulting in economic loss of the ruminant industry worldwide. In comparison to the usage of anthelmintic reagents which can cause resistance among parasitic nematodes, vaccination represents a sustainable and effective approach. Vaccination with a mixture of native Haemonchus gut glycoproteins (namely H11 antigens) has shown effective protection in lambs. However, attempts to produce these antigens recombinantly in various expression hosts and use them as vaccines resulted in either low or no protection in animal trials. As the native H11 antigens are known to carry additional modifications with sugars on the surface of proteins (termed “glycosylation”), which plays an important role in the biological function of antigens, investigation on how to mimic these natural sugar modifications on the recombinant H11 became necessary and will facilitate the production of effective antigens. The sugar structures on the native H11 antigens possess a special “core” modified with up to three fucose residues, which are enzymatically synthesised by three fucosyltransferases. A commercial insect cell line (Hi5) is a suitable host for glycoengineering using recombinant baculovirus to introduce genes encoding Caenorhabditis elegans glyco-enzymes, which can be used to effectively remodel the sugars on the selected helminth reporter. Full length nematode glyco-enzyme encoding sequences are sufficient for Golgi targeting and realising glycoengineering in insect cells. The biological role of C. elegans glyco-enzymes will be investigated by studying the N-glycomes of relevant mutants using HPLC and mass spectrometers as major tools. Recombinant baculoviruses carrying genes encoding two C. elegans glyco-enzymes and DNA sequences encoding Haemonchus H11 (expression targets) will be prepared using molecular biology approaches to produce H11 antigens in Hi5 insect cells. In addition to the protein sequences and peptidase activities, the biochemical properties of glyco-engineered H11 antigens will be evaluated focusing on the sugar modifications and other potential protein modifications. This is probably the first attempt to express helminth antigens modified with authentic sugars in insect cells. The glyco-engineered recombinant H11 antigens are expected to better mimic the native H11 antigens; therefore they may serve as a vaccine candidate in animal trials to protect ruminants from Haemonchus infection.

Supervised Theses and Dissertations