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. These master projects are in the field of biochemistry and structural biology.
Background about thiols: 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 Staphylococcus aureus and Bacillus cereus, use the unique bacterial LMW thiol bacillithiol (BSH). We have solved the structure of the bacillithiol disulfide reductase (Bdr) enzyme that regenerates BSH after it has been used as an antioxidant. Another important role of BSH is thiol-protection under oxidative stress, through the redox post-translational modifications (PTM) termed protein S-bacillithiolation of Cys residues avoiding overoxidation and inactivation. To regenerate the active enzymes after stress, de-bacillithiolation is needed. This is catalyzed by the bacilliredoxin (Brx) redox pathway. Brxs attack the active site Cys on the BSH-mixed protein disulfide on S-bacillithiolated substrates, resulting in the transfer of BSH to the Brx active site Cys. Currently, three Brxs have been identified, which seem to have different specificity towards different target enzymes. However, the diversity among the Brxs and in protein substrates is not established. The Brx-SSB intermediate can then be reduced by BSH, leading to the oxidised product BSSB, which again is reduced by Bdr.
Here master projects would focus on understanding the diversity among the Brxs, and investigate Brxs as potential drug targets. This would involve solving structures of the three Brxs with and without bound BSH, determination of properties like pKa to distighuish the isoforms and to look on binding of potential drug fragments. Complex structures between Brx and target protein would be beneficial.
Background about metals: For these bacteria to grow and survive during infection, they need access to essential metals like iron. These bacterial 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 that can provide the electrons needed from NADPH. We have previously solved the structure of one of these HOs from Bacillus cereus, however, there are also two other potential HOs in this bacteria, and the efficiency and diversity of these enzymes are lacking.
Here master project would focus on understanding the diversity among the HOs, and investigate them as a potential drug target. This would involve solving structures of the HOs with and without bound heme, determining the kinetics for the isoforms and to look on binding of potential drug fragments. Complex structure between reductase and HO would be beneficial.
To be able to answer these questions 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 supervisors.
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 project will be performed in the Structural Redox Enzymology - Hersleth Lab (Section for Biochemistry and Molecular Biology) and supervised by Hans-Petter Hersleth and Marta Hammerstad.
Contact: Hans-Petter Hersleth room 2313, e-mail: h.p.hersleth@ibv.uio.no
To read more see our group homepage: http://hersleth.org/
Some relevant references:
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. [Link]
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. [Link]
M. Hammerstad & H.-P. Hersleth
Overview of structurally homologous flavoprotein oxidoreductases containing the low Mr thioredoxin reductase-like fold – A functionally diverse group.
Arch. Biochem. Biophys. (2021), 702, 108826. [Link]