Master projects will focus on deciphering important enzymes in these unique bacterial redox defense and survival networks. The growing antimicrobial resistance evolving among pathogens increases the need to search for new antimicrobial targets, including enzymatic networks involved in the maintenance of pathogens redox and metal homeostasis as well as the deoxyribonucleotide pool. The master project is in the field of biochemistry and structural biology, and can focus on either of these three directions.
Background and projects
Project about flavins and deoxyribonucleotides
The last step in the synthesis of the DNA building blocks in all organisms involve the reduction of ribonucleotides to deoxyribonucleotides by ribonucleotide reductase (RNR). In humans the RNR contain a di-iron cluster while many bacteria use instead an isoform of RNR containing a di-manganese cluster. For this bacterial RNR to be active in producing deoxyribonucleotides it needs a flavin activase containing FMN, which again needs to obtain electrons from a NADPH dependent FAD containing reductase (FNR). The potential reductases for NrdI in Bacillus cereus have been assessed, however, in Staphylococcus aureus this is lacking
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Here a master project would focus both on discovery of antimicrobial drug candidates against NrdI and understanding the function between NrdI and its reductase. For discovery of drug candidates against NrdI a fragment-based crystallographic approach will be used by soaking ligands into NrdI crystals and solving the structures to identify potential candidates. To compare the function and interaction of NrdI with its reductase in Bacillus cereus and Staphylococcus aures kinetic studies will be performed. Binding between NrdI and the reductase will be investigated by biophysical methods and by docking of NrdI to the reductase.
Project about metals
For these bacteria to grow and survive during infection, they need access to essential metals like iron. These bacteria have therefore systems to acquire iron from the infected host, either by small chelating molecules called siderophores that can capture iron and transport into the bacteria, or through capturing heme from the hosts hemoglobin, transporting it into the bacteria, and there use heme oxygenases (HO) that break down heme groups and release iron. Pathogenic bacteria have specific HOs that can break down heme and release iron. To do this they need to be activated by a reductase enzyme (iron-utilization oxidoreductase) that can provide the electrons needed from NADPH. In Bacillus cereus there are three potential HOs and understanding of the efficiency and diversity of these enzymes are lacking.
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Here a master project would focus on understanding the diversity among the HOs. This could involve solving structures of HOs with and without bound heme, structure of reductase with and without bound NAPDH, determining the kinetics for the isoforms, difference in binding between the reductase and HOs, docking of reductase and HOs and to look on binding of potential drug fragments.
Project about thiols
Low-molecular weight (LMW) thiols are a class of antioxidant molecules involved in many important cellular processes in all organisms, including a critical protective role in cells by maintaining cytosolic proteins in their reduced state to maintain the redox homeostasis. Humans use the LMW thiol glutathione, while important pathogens like the Bacillota bacteria Staphylococcus aureus and Bacillus cereus, use the unique bacterial LMW thiol bacillithiol (BSH). as a defense mechanism to buffer the intracellular redox environment and counteract oxidative stress encountered by human neutrophils during infections. In this defense BSH is scarified and oxidised to BSSB (bacillithiol disulfide), however, regenerated by the bacillithiol disulfide reductase (Bdr) enzyme. BSH and Bdr has been proposed in several other bacteria as Acidobacteriota, Bacteriodota and Deinococcota.
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Here a master project would focus on understanding the diversity of the Bdrs among different type of bacteria. This would involve solving structures of Bdrs from different bacteria, structure with bound BSH and NAPDH and investigate the activity of BSSB reduction by the different Bdrs.
Methods
For these master projects you will need to express and purify the proteins, solve the structure of them and/or do biochemical and biophysical characterisation. There are several different possibilities in master projects within these topics.
Examples of methods you normally will use and learn:
- Protein expression
- Protein purification
- Protein crystallisation
- Solving the crystal structure of proteins
- Activity studies
- Enzyme kinetics
- Anaerobic work
- Spectroscopic charaterisation
- Binding studies
Data collection for solving the structures will involve travelling abroad to synchrotrons. The biophysical and biochemical methods used in this master project will to a large extend be covered in BIOS4020, taught by the supervisor.
Through the master project, you will also learn to
- Present your work orally through group meetings
- Present your work through posters presentations at scientific conferences.
- Learn to plan and perform scientific work
- Learn to write up your work as a thesis
Supervision
The master projects will be performed in the Structural Redox Enzymology - Hersleth lab (Section for Biochemistry and Molecular Biology) and in the Structural biology core facility (PX-Oslo) and supervised by Hans-Petter Hersleth and co-supervised by Bjørn Dalhus.
Contact: Hans-Petter Hersleth room 047S016 (Life Science Building), e-mail: h.p.hersleth@ibv.uio.no
To read more see our group homepage: http://hersleth.org/
Some relevant references:
M. Lofstad, I. Gudim, M. Hammerstad, Å.K. Røhr & H.-P. Hersleth
Activation of the Class Ib Ribonucleotide Reductase by a Flavodoxin Reductase in Bacillus cereus.
Biochemistry (2016), 55, 4998-5001.M. Hammerstad, A,K. Rugtveit, S. Dahlen, H.K. Andersen & H.-P. Hersleth
Functional Diversity of Homologous Oxidoreductases—Tuning of Substrate Specificity by a FAD-Stacking Residue for Iron Acquisition and Flavodoxin Reduction.
Antioxidants (2023), 12, 1224.M. Hammerstad, I. Gudim & H.-P. Hersleth
The Crystal Structures of Bacillithiol Disulfide Reductase Bdr (YpdA) Provide Structural and Functional Insight into a New Type of FAD-Containing NADPH-Dependent Oxidoreductase.
Biochemistry (2020), 59, 4793-4798.