Projects/Interests

The Orange Protein Complex

PhD Project 7, PTNMRPhD

    An orange-colored protein (ORP) was isolated from a sulphate reducing bacterium, Desulfovibrio species, grown in a lactate-sulphate medium under anaerobic conditions. This protein is a monomer of 11.8 kDa and contains a novel mixed-metal sulphide cluster of the type [S2MoS2CuS2MoS2]3- (Figure 1), which is non-covalently bound to the polypeptide chain [1,2].
The ORP protein has a sequence homology of around 30-40 % to conserved proteins from eubacteria and hyperthermophilic archaea with unknown function, which have a conserved structural domain common to the nitrogenase accessory factors (NifB and NifX) [3].
   


References

1. S.R. Pauleta*, et al., Biomolecular NMR Assignments, 2007, 1, 81-3.

2. A. Fiévet, L. My, E. Cascales, M. Ansaldi, S. R. Pauleta, I. Moura, Z. Dermoun, C.S. Bernard, A. Dolla, C. Aubert. The anaerobe-specific ORP complex of Desulfovibrio vulgaris Hildenborough is encoded by two divergent operons co-regulated by 54 and a cognate transcriptional regulator. J. Bacteriology. 2011. 193(13):3207-19.

3. R. Grazina, S.R. Pauleta*, J.J.G. Moura, I. Moura. 3.06 - Iron-sulfur centers: new roles for ancient metal sites. In "Comprehensive Inorganic Chemistry II". Vol. 3: Bioinorganic Fundamentals and Applications: Metals in Natural Living Systems and Metals in Toxicology and Medicine. Ed. Vincent Pecoraro. 2013. page 103-48. Elsevier. ISBN: 9780080977744.

4. M.S.P. Carepo, S. R. Pauleta*, A.G. Wedd, J.J.G. Moura, I. Moura. Mo-Cu metal cluster formation and binding in an Orange Protein isolated from Desulfovibrio gigas. 2014. J. Biol. Inorg. Chem. 19(4-5):605-14.

5. M.S. Carepo, C. Carreira, R. Grazina, M.E. Zakrzewska, A. Dolla, C. Aubert, S. R. Pauleta, J.J.G. Moura, I. Moura. Orange protein from Desulfovibrio alaskensis G20: insights into the Mo-Cu cluster protein-assisted synthesis. 2016. J. Biol. Inorg. Chem. 21(1):53-62.

6.A. Neca, R. Soares, M.S.P. Carepo, S. R. Pauleta*. Resonance assignment of DVU2108 that is part of the Orange Protein complex in Desulfovibrio vulgaris Hildenborough. 2016. Biomol. NMR Assign. 10:117-20.

7. B.K. Maiti, I. Moura, J.J.G. Moura, S.R. Pauleta*. The Small Iron-Sulfur Protein from the ORP operon binds a [2Fe-2S] cluster". 2016. Biochim. Biophys. Acta. 1857(9):1422-9.

8. B.K Maiti, L.B. Maia, S.R. Pauleta, I. Moura, J.J.G. Moura. 2017. Inorg. Chem. DOI: 10.1021/acs.inorgchem.6b02906. Epub Jan 27 2017.


Oxidative Stress Response - Bacterial Cytochrome c Peroxidase

Reactive oxygen species (ROS) (e.g., hydrogen peroxide, superoxide anion) are produced by the immune system to kill invading microorganims. ROS are potent cytotoxic oxidants that can cause damage in the DNA, proteins and lipids, leading to cell damage or death, properties that are exploit by the immune system. Hence, the ability to combat these toxic compounds is fundamental for bacteria survival in the host. Indeed, bacteria have developed a notable array of enzymatic systems for their detoxification, such as superoxide dismutases, catalases and peroxidases.

Recently, it has been shown that cytochrome c peroxidase (CCP) plays a key role in virulence, in pathogenic bacteria, by protecting these microorganims against ROS. In these bacteria, the gene coding for CCP is under the regulation of OxyR, a transcription regulator that responds to increased levels of hydrogen peroxide. In addition, strains of Neisseria with a knock-out in their oxyR or/and ccp gene were shown to be more susceptible to hydrogen peroxide and less virulent.

The CCPs are di-haem c-type enzymes that catalyze the reduction of hydrogen peroxide to water. It is our purpose to isolate the CCPs from the pathogenic bacteria Neisseria gonorrhoeae, as well as the homologous enzyme from Escherichia coli.

Reactive oxygen species like superoxide radical (O2·-), hydrogen peroxide (H2O2), and hydroxyl radical (HO·) are toxic compounds produced by the incomplete reduction of oxygen during oxidative metabolism. These compounds are cytotoxic oxidants that can initiate lipid peroxidation, cause DNA strand breaks and indiscriminately oxidise organic molecules, leading to cell damage or . These reactive oxygen species are also thought to be important in the aging process and in the pathogenesis of neurodegenerative diseases such as Parkinson’s disease.

The dihaem bacterial cytochrome c peroxidase isolated from Paracoccus pantotrophus is a periplasmic enzyme that reduces peroxide to water using the mono-haem c-type cytochromes and/or type I copper protein, pseudoazurin as electron donors.


Novel Bacterial System Involved in Copper Tolerance

Copper is an essential trace element required by all aerobic organisms. Copper ions are used as a structural and/or catalytic cofactor in several enzymes (e.g., cytochrome c oxidase and superoxide dismutase). However, free copper ions are highly toxic due to their ability to generate reactive oxygen species, via Fenton-type reactions, which damage lipids, DNA and proteins.

It is crucial for any organism to maintain a tight regulation of the intracellular concentrations of copper ions, a role played by the copper homeostasis systems. Nevertheless, in bacteria as well as in eukaryotic organisms, the molecular mechanisms essential for copper homeostasis are only partially understood. Bacterial systems have been considered as models systems to investigate the mechanism of copper homeostasis, due to their simplicity and easy gene manipulation. In fact, bacterial efflux copper pumps and chaperons are similar to their eukaryotic counterparts.

The study of bacterial copper homeostasis, especially copper tolerance mechanisms, has gain interest due to the widespread use of copper compounds as bactericide in agriculture or as disinfectants in the food industry. This general use will undoubtedly lead to the selection of copper-resistance strains of bacteria and the dissemination of plasmid-borne copper resistance genes among different bacterial pathogens with important implications in disease control efforts. Moreover, mechanisms of copper tolerance have recently been linked to the molecular mechanism of pathogenesis. During infection, bacteria are exposed to different stresses, as reactive oxygen species and fluctuations in metal ions concentration. Indeed, phagocytes infected with Mycobacterium and Salmonella present increased copper levels, that for Mycobacterium tuberculosis, showed to specifically induce the transcription of a gene encoding for a P-type ATPase copper efflux pump. Therefore, there is an increased urge to understand the different bacterial mechanisms of copper tolerance in order to develop new therapeutic targets and improve current copper-based strategies.

Under the scope of copper resistance, we will investigate the molecular mechanism of copper resistance in a bacterial system, Marinobacter hydrocarbonoclasticus, which presents some unique features.



Characterization of Transient Protein Interaction

Under Construction.