Structural biology: Flavin-based enzyme activation in bacteria - finding redox partners
Basic understanding of how enzymes work and are activated is important with respect to both medical and industrial applications. A central class of enzymes is redox enzymes using redox cofactors for electron transfer. Many enzymes need to be activated to perform their catalytic functions. Such activating networks in many bacteria involve different flavin proteins. In such networks, NADPH is used as a primary electron donor for different flavin-based reductases, which in turn deliver electrons to different electron transfer proteins, which again activate essential enzymes like ribonucleotide reductase and nitric oxide synthase. The focus of this master project will be to decipher flavin-based redox networks, and will be in the field of biochemistry and structural biology.
Two of the enzyme systems that are activated by flavin-based redox networks are some classes of ribonucleotide reductase (RNR, an enzyme found in all organism being the only enzyme that converts the ribonucleotides to deoxyribonucleotides), and nitric oxide synthase (NOS, the enzyme that generate the both signaling and toxic molecule NO). In the firmicutes bacteria (including pathogens as Bacillus anthracisand Staphylococcus aureus) the flavin-based activation are performed by a more recently discovered class of reductases. Since this is different from the mammalian types as well as other bacteria, understanding this activation network could be a potential point of attack for antibiotics against these pathogens.
In Bacillus cereus there are actually 3 flavodoxins (Flds) and 2 ferredoxins (Fds) that could receive electrons from 3 ferredoxin/flavodoxin reductases (FNR) and further deliver them to the enzymes RNR or NOS. We have recently shown that the FNR2 is the workhorse enzyme with respect to the Flds in B. cereus, and identified a key residue in the active site of FNR2 that differs from FNR1. We would therefore like to make the cross-mutation of this residue between FNR1 and FNR2 to se how this influences the electron transfer rates, and to prove that this residue is essential for the distinction between FNR1 and FNR2. The FNR1 also have an additional C-terminal helix that we want to remove to see the effect this has on the reduction rates since it is believed to be involved in substrate recognition. Since the FNR1 has a lower turnover with respect to the Flds, we would like to investigate if iron-sulfur Fds could be the superior redox partner for FNR1, especially since the firmicute S. aureuslack both the FNR2 and Flds, but contain a FNR1 variant and a Fd.
In humans the ribonucleotide reductase RNR is activated by a thioredoxin network and a di-iron protein. However, in bacteria this picture is more complicated because different bacteria have adopted to alternative activation routes to counteract human innate immunity that tries to kill the bacteria by creating e.g. iron limited environments. Therefore, bacterial classes that use di-manganese enzymes and flavodoxin activation networks have evolved. Last year a class that is able to perform the RNR activation without use of metals but only a flavin activation network has been discovered. How, the flavin network works here is not known, and need to be elucidated.
Research projects will focus on understanding these electron transfer processes on a molecular level, their selectivity for redox partners, and their electron transfer efficiency in activating their redox partners. How these redox partner proteins interacts is also lacking, so trying to crystallise protein-protein complexes would also be a highly desired goal. To be able to answer these questions you will need to express and purify the proteins, solve the structure of them and do some biochemical and biophysical characterisation.
There are several different possibilities in master projects within these topics, depending on the interest of the master student. Below are some possibilities listed:
Possibility 1 will be to express, purify and crystallise/solving structure of the FNR1 mutant, FNR2 mutant and the truncated FNR1 from B. cereus, determine their redox potentials and their efficiency in reducing the Flds.
Possibility 2 will be to express, purify and crystallise/solving structure of the proteins in the activation pathway of the metal free RNR class, identify the redox partners, determine their redox potentials and their efficiency in reducing the Flds/RNRs.
Methods you will use and learn:
- Protein expression
- Protein purification
- Protein crystallisation
- Solving the crystal structure of proteins
- Enzyme kinetics
- Redox potential measurements
- Anaerobic work
- Spectroscopic charaterisation.
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 Biochemistry - Hersleth Group (Section for Biochemistry and Molecular Biology) and supervised by Hans-Petter Hersleth and Marta Hammerstad.
Contact: Hans-Petter Hersleth room 2313, e-mail: firstname.lastname@example.org
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. [Link]
M. Hammerstad, H.-P. Hersleth, A.B. Tomter, Å.K. Røhr & K.K. Andersson
Crystal Structure of Bacillus cereus Class Ib Ribonucleotide Reductase Di-iron NrdF in Complex with NrdI.
ACS Chem. Biol. (2014), 9, 526-537. [Link]
I. Gudim, M. Lofstad, W. van Beek & H.-P. Hersleth
High-resolution crystal structures reveal a mixture of conformers of the Gly61-Asp62 peptide bond in an oxidised flavodoxin from Bacillus cereus.
Protein Sci. (2018), 27, In Press. [Link]