MASTER EXAMS
10:15: Marie Lofstad
Tid og sted: 27. jun. 2012 10:15, Rom 2203 Kristine Bonnevies hus
The nitric oxide synthase protein system in Bacillus cereus - From gene to 3D structure
Nitric Oxide Synthase (NOS) is the enzyme that produces nitric oxide in the body from arginine and oxygen. In mammals three different NOS isoforms with different functions exist: endothelial eNOS, neuronal nNOS and inducible iNOS. The mammalian NOS consists of one oxygenase domain (with heme/biopterin), one reductase domain (with FMN/FAD) and a calmodulin binding domain. The reductase domain transports electrons to the heme. Bacterial NOSs, on the other hand, consist only of an oxygenase domain, and separate proteins deliver the electrons to this domain.
The possible functions of bacterial NOSs are not fully understood. We have studied a NOS-like protein from Bacillus cereus, BC5444, which is of interest since it is a close relative to the pathogen Bacillus anthracis. Like other bacterial NOSs, it is lacking a reductase domain; however, several possible reductase partners exist in the genome. Flavodoxins are most likely candidates, due to their capacity to deliver electrons through their co-factor flavin mononucleotide (FMN). Two such flavodoxins, BC1376 and BC3541, have been further investigated.
How electrons are transferred by these two flavodoxins from a flavodoxin reductase to NOS, is currently not known. Another intriguing question concerns the specificity of the flavodoxins – is one specific flavodoxin a more efficient electron donor for NOS than the other, or are they equally good?
Different biochemical and structural techniques are applied in order to try to answer these questions. So far BC5444, BC3541 and BC1376 have been cloned, expressed, purified and crystallized, and the structure of the flavodoxin BC1365 has been solved at high resolution of 1Å using X-ray crystallography. Crystals of the flavodoxin BC3541 and the NOS BC5444 have diffracted to approximately 3Å.
13:15 Silje Skråmo
Studies of Electron Transfer from Flavodoxin Reductase to NrdI in Bacillus cereus
The Ribonucleotide reductase (RNR) catalyzes the reduction of nucleoside di- or triphosphates to deoxynucleoside di- or triphosphates in all organisms and play a central role in nucleic acid metabolism. The reducing equivalent is delivered to the RNR through a series of redox proteins. The aim of the project is to study if and how the flavodoxin reductases (FldRs) BC4926 and BC0385, found in Bacillus cereus, transfer electrons via the flavodoxin NrdI to the RNR system in this organism. This will be carried out through structure determination via x-ray diffraction experiments and activity measurements.
The function of NrdI in the class Ib RNR system is yet under investigation. The two FldRs found in Bacillus cereus are candidates for transferring electrons from NADPH to NrdI, which in turn forms a complex with the NrdF subunit of RNR. The function of NrdI, as well as the whole system, might therefore be better understood by studying the FldRs. Moreover, a suggestion of which reductase that works best in vitro, and maybe also in vivo, will make more detailed studies of the interaction between NrdI and NrdF, as well as the generation of the metal cluster-tyrosyl radical cofactor, possible.
The FldR gene BC0385 has been cloned into a pET-22b expression vector, and purification of this protein as well as the other FldR and NrdI has been carried out. Initial crystals of the FldR BC4926 have been obtained for both the apo and holo form of the protein. Activity measurements are in progress, and are so far indicating that both FldRs are reduced by both NADH and NADPH.