Chemistry of enzymes and antimicrobial proteins subproject

subproject leader: Dr. Gyöngyi Gyémánt

I. Structure of the subproject

 1. Glycoenzyme WG

           group leader: Dr. Gyöngyi Gyémánt (http://www.inorg.unideb.hu/oktatok/41).      

2. Antimicrobial proteins and peptides WG

           group leader: Prof. Dr. Gyula Batta (http://www.structbiol.unideb.hu/)

3. D-galactose WG

           group leader: Dr. Levente Karaffa (http://biochemeng.unideb.hu/)

II. Scientific goals

1. Research on human glycoenzymes

Enzymes in polysaccharide degradation and conversion with the aim of dissecting metabolic pathways.

Chemoenzymatic synthesis of oligosaccharides.

2. Structure determination of new antifungal proteins (PAFB, NFAP2, NFAP)

In-vitro investigation of the interactions of antifungal proteins with sensitive filamentous fungi and model systems

Structure determination of modified glycopeptide antibiotics

Investigation of carbohydrate-protein interactions using NMR and other biophysical methods (ITC, SPR)

3. Understanding the molecular mechanisms of D-galactose assimilation in the model fungus Aspergillus nidulans

Identification of the structural and regulatory components through the characterization of mutant strains defective in lactose and/or D-galactose metabolism.

Elucidating the role cytosolic citrate synthases play in the overproduction of citric acid in A. niger.

Identification of the putative cytosolic citrate synthase orthologs in A. terreus.

III. Expected results

1. Characterisation of glycoenzyme targets involved in metabolic syndrome.

Identification of pharmaceutically active ingredients in functional food and dietary supplements for the prevention and treatment of diabetes and obesity.

Preparation of prebiotic oligosaccharides from plant and fungal polysaccharides.

2. Deposition of the structures of the new antifungal proteins into the PDB data base.

Disclosing and better understanding of the mode of action of the new antifungal protein agents.

Structural characterisation of glycopeptides with novel action profile and new mode of action.

Design of new heparine derivatives possessingbeneficial effect against blood coagulation, based on structure-dynamics-biological relationship analysis.

Design and characterisation of new carbohydrate derivatives that are able to influence the mode of action of biologically important lectin proteins.

3. Quantitative assessment of the distribution of carbon flux between the Leloir and the oxido-reductive pathways of fungal D-galactose catabolism, using the model fungus A. nidulans.

Conclusive (experimental) evidence on the dispensability of mitochondria during citrate biosynthesis in A. niger.

In silico evidence on the dispensability of mitochondria during itaconate biosynthesis in A. terreus.

IV. Infrastructure

Instruments to be purchased within the framework of the project:

HPLC (assembled with Corona CAD detector):

The application of the HPLC technique is essential to analyze the products of the enzymatic reactions and the components of plant extracts.  The analysis, which can be performed by using two different kind of detectors (DAD and Corona CAD), depends on the chemical nature of the sample components. Gradient elution is often used when the separation of the substances vary greatly in size and polarity. Following the enzymatic reactions over time requires serial measurements by the use of thermostat controlled autosampler.

Vacuum centrifuge and lyophiliser system

The vacuum centrifuge and lyophiliser makes an ideal system for the concentration of biological or heat sensitive samples such as proteins and metabolites. The freeze-dry technique is applied when enzyme sample preparations are needed for long-term strorage or for the isolation of heat sensitive synthetic, or natural compound from  aqueous solvent. The vacuum centrifuge uses the combination of centrifugal force, vacuum and heat for the speed evaporation of organic solvent from multiple small samples, including HPLC purified and synthesized substances.

DSC microcalorimeter

The differential scanning calorimetry and the isothermal titration calorimetry are complementary analytical tools for characterizing the stability and the conformational changes of macromolecules including proteins. They are also valuable techniques at characterizing the interactions of biomolecules without labelling requirements. Applications range from confirming intended binding targets in small molecule drug discovery to the development of stable biotherapeutics. DSC is particularly useful for characterizing very tight or slow binding interactions. At the moment one of our labs is equipped with ITC and DCS is under purchase.

Helium compressor

To be used for the recovery of evaporated helium gas from our superconducting NMR spectrometers. Application will reduce the running costs significantly.

HPLC (with Open Lab drive)

The equipment is needed for two major purposes: (1) kinetic analysis: to set up time-profiles for the concentration changes of residual carbon sources (e.g., lactose, D-galactose, D-glucose) during batch fermentations, and (2) to determine the concentration of intracellular metabolites related to D-galactose assimilation in fungi, such as D-galactose-1-phosphate or galactitol. Refractive Index (RI)-based detection is the generally used method of choice for both groups of compounds.