Periodic Reporting for period 2 - TIMB3 (Twin to Illuminate Metals in Biology and Biocatalysis through Biospectroscopy)
Okres sprawozdawczy: 2019-12-01 do 2022-06-30
Metals play essential roles in life. Just think of iron in blood or vitamin B12. Nevertheless, a deeper understanding of mechanisms of action of metals in metalloproteins can radically transform our capacity to improve health and well-being and lead to novel biotechnological solutions for pressing environmental problems.
TIMB3 performed innovative research on metalloproteins using biospectroscopy, providing detailed molecular insights into the role of diverse metals in biology. The key target systems were metal homeostasis and trafficking in living cells, biogenesis of essential metallocofactors, and biocatalysis by metalloenzymes. Besides the host institution, ITQB NOVA, TIMB3 involved two partners, CIRMMP (Italy) and TUB (Germany), which possess unmatched expertise in Magnetic Resonance and Vibrational spectroscopies. The project envisaged scientific exchange missions among the partners, and training events on scientific and soft skills. Furthermore, ITQB NOVA created a Virtual Platform for Biospectroscopy to be used by non-specialists, from which they can learn about the potential of this versatile group of methods and its applicability to their specific research problems, broadening the social awareness of the capacities of biospectroscopy.
The overall objective of the TIMB3 project, to increase the excellence of ITQB NOVA to reach an international level in the field of biospectroscopy applied to metalloproteins, has been reached, through scientific achievements, improving the scientific visibility of ITQB NOVA, the involvement in European projects and enhancing the critical mass of researchers in the biospectroscopy field. The work developed contribute to the overall objective of the TIMB3, providing new scientific insights into challenging topics beyond the state of the art, which are expected to have socio-economic impact.
TIMB3 performed innovative research on metalloproteins using biospectroscopy, providing detailed molecular insights into the role of diverse metals in biology. The key target systems were metal homeostasis and trafficking in living cells, biogenesis of essential metallocofactors, and biocatalysis by metalloenzymes. Besides the host institution, ITQB NOVA, TIMB3 involved two partners, CIRMMP (Italy) and TUB (Germany), which possess unmatched expertise in Magnetic Resonance and Vibrational spectroscopies. The project envisaged scientific exchange missions among the partners, and training events on scientific and soft skills. Furthermore, ITQB NOVA created a Virtual Platform for Biospectroscopy to be used by non-specialists, from which they can learn about the potential of this versatile group of methods and its applicability to their specific research problems, broadening the social awareness of the capacities of biospectroscopy.
The overall objective of the TIMB3 project, to increase the excellence of ITQB NOVA to reach an international level in the field of biospectroscopy applied to metalloproteins, has been reached, through scientific achievements, improving the scientific visibility of ITQB NOVA, the involvement in European projects and enhancing the critical mass of researchers in the biospectroscopy field. The work developed contribute to the overall objective of the TIMB3, providing new scientific insights into challenging topics beyond the state of the art, which are expected to have socio-economic impact.
Herein we highlight the most relevant scientific achievements,
i) Biosensor construction: The results obtained provide a proof of concept and will lead to a mediator-less 3rd generation biosensor for the detection of hydrogen peroxide (H2O2). We are close to the development of such a biosensor in disposable, miniaturized, single-use configuration. Detection and quantification of H2O2 is relevant in pharmaceutical, environmental, textile, paper, food and chemical industries, in which it is used as an oxidant for whitening or sterilizing purposes in medicine, as it plays the roles of oxidative stress marker in aging and disease, and of defence agent in response to pathogens. We employed immobilized DyP type peroxidases and their variants, which show superior sensitivity and shorter response times than any other enzyme based H2O2 biosensor reported so far in the literature. Furthermore low-cost H2O2 sensors are highly sought after for numerous applications.
ii) Improved DNA repair in humans: bacterial and human DNA endonucleases research provides new insights into the mechanistic properties of these enzymes, and understanding of the search for and repair of damaged DNA. The obtained results will be exploited in two different manners: i) interfere with DNA repair in pathogens and ii) improve DNA repair in humans, which will have an impact on health and aging-related societal challenges.This is particularly relevant in the case of human endonuclease, hNTH1, crucial for genome maintenance in humans, as the mutations in the nth1 gene are associated with development of adenomatous polyposis and colorectal cancer.
iii) Development of new NMR methodologies: New NMR methods were developed in executed missions, which are/will be made freely available to the spectroscopy community via publications. These methods are specifically tailored for fast-relaxing systems such as metalloproteins and enzymes. These constitute approximately 30% of the proteins encoded in the genomes and are the focus of research on many fronts, as biotechnology for novel industrial production methods, or biochemistry for the identification of drug targets in infectious or degenerative diseases. Furthermore, the missions performed showed the potential of paramagnetic restraints to upgrade the current paradigm for the determination of solution structures of biological macromolecules by NMR. Broadening the range of procedures that can inform on the structure and dynamics of macromolecules will increase the options for performing this characterization in conditions that are biologically relevant.
iv) Alternative diagnostic tools: RT-LAMP is a strong alternative for diagnosis of infections caused by SARS-CoV-2, based on viral genetic material detection by the color change of a pH-sensitive dye. In order to use saliva samples and bypass a potential bias originating from often acidic saliva, we developed a new colorimetric pH-independent alternative, based on the divalent zinc salt and the complexometric indicator murexide. The developed methodology can revolutionize SARS-Cov-2 detection (patent filed).
v) Revealing the antifungal synergism between copper and fluconazole: Copper potentiates the activity of the antifungal fluconazole in C.glabrata an opportunistic yeast intrinsically tolerant to the drug and often associated with life-threatening Candida infections. We revealed that copper promotes the accumulation of fluconazole within the fungus, which amplifies the drug efficiency, which led us to synthetize novel promising antifungal copper-containing molecules.
vi) Characterization of catalytic intermediate species and processes:
Hydrogenases: studies with HysAB from Desulfovibrio vulgaris Hildenborough elucidated the catalytic intermediates and non-catalytic states of this NiFeSe hydrogenase, interesting from a biotechnological point of view as it has a high activity and is resistant to oxygen inactivation. We provided a better understanding of the enzyme and its mechanism, namely for O2 tolerance, as well as H2 reduction and oxidation.
Flavodiiron nitric oxide reductases: to characterize flavodiiron nitric oxide (NO) reductases active site, the bacterial E.coli enzyme was studied by Nuclear Vibrational Resonance Spectroscopy, to unravel the structure of the catalytic center. Further insights of the catalytic center will be used to tailor new synthetic catalysts.
Iron-Sulfur Cluster biogenesis and repair: we obtained novel insights into the structural basis of iron donation for FeS cluster biogenesis and novel FeS repair proteins have been identified. This increased our understanding of the processes that involve FeS cluster containing proteins and advanced the research that focuses on these systems.
The results obtained, have been disseminated at international events and meetings: around 80 participations of TIMB3 members at events across the globe, including Europe, USA, Canada and China, which took place in person or in virtual/hybrid modes.
i) Biosensor construction: The results obtained provide a proof of concept and will lead to a mediator-less 3rd generation biosensor for the detection of hydrogen peroxide (H2O2). We are close to the development of such a biosensor in disposable, miniaturized, single-use configuration. Detection and quantification of H2O2 is relevant in pharmaceutical, environmental, textile, paper, food and chemical industries, in which it is used as an oxidant for whitening or sterilizing purposes in medicine, as it plays the roles of oxidative stress marker in aging and disease, and of defence agent in response to pathogens. We employed immobilized DyP type peroxidases and their variants, which show superior sensitivity and shorter response times than any other enzyme based H2O2 biosensor reported so far in the literature. Furthermore low-cost H2O2 sensors are highly sought after for numerous applications.
ii) Improved DNA repair in humans: bacterial and human DNA endonucleases research provides new insights into the mechanistic properties of these enzymes, and understanding of the search for and repair of damaged DNA. The obtained results will be exploited in two different manners: i) interfere with DNA repair in pathogens and ii) improve DNA repair in humans, which will have an impact on health and aging-related societal challenges.This is particularly relevant in the case of human endonuclease, hNTH1, crucial for genome maintenance in humans, as the mutations in the nth1 gene are associated with development of adenomatous polyposis and colorectal cancer.
iii) Development of new NMR methodologies: New NMR methods were developed in executed missions, which are/will be made freely available to the spectroscopy community via publications. These methods are specifically tailored for fast-relaxing systems such as metalloproteins and enzymes. These constitute approximately 30% of the proteins encoded in the genomes and are the focus of research on many fronts, as biotechnology for novel industrial production methods, or biochemistry for the identification of drug targets in infectious or degenerative diseases. Furthermore, the missions performed showed the potential of paramagnetic restraints to upgrade the current paradigm for the determination of solution structures of biological macromolecules by NMR. Broadening the range of procedures that can inform on the structure and dynamics of macromolecules will increase the options for performing this characterization in conditions that are biologically relevant.
iv) Alternative diagnostic tools: RT-LAMP is a strong alternative for diagnosis of infections caused by SARS-CoV-2, based on viral genetic material detection by the color change of a pH-sensitive dye. In order to use saliva samples and bypass a potential bias originating from often acidic saliva, we developed a new colorimetric pH-independent alternative, based on the divalent zinc salt and the complexometric indicator murexide. The developed methodology can revolutionize SARS-Cov-2 detection (patent filed).
v) Revealing the antifungal synergism between copper and fluconazole: Copper potentiates the activity of the antifungal fluconazole in C.glabrata an opportunistic yeast intrinsically tolerant to the drug and often associated with life-threatening Candida infections. We revealed that copper promotes the accumulation of fluconazole within the fungus, which amplifies the drug efficiency, which led us to synthetize novel promising antifungal copper-containing molecules.
vi) Characterization of catalytic intermediate species and processes:
Hydrogenases: studies with HysAB from Desulfovibrio vulgaris Hildenborough elucidated the catalytic intermediates and non-catalytic states of this NiFeSe hydrogenase, interesting from a biotechnological point of view as it has a high activity and is resistant to oxygen inactivation. We provided a better understanding of the enzyme and its mechanism, namely for O2 tolerance, as well as H2 reduction and oxidation.
Flavodiiron nitric oxide reductases: to characterize flavodiiron nitric oxide (NO) reductases active site, the bacterial E.coli enzyme was studied by Nuclear Vibrational Resonance Spectroscopy, to unravel the structure of the catalytic center. Further insights of the catalytic center will be used to tailor new synthetic catalysts.
Iron-Sulfur Cluster biogenesis and repair: we obtained novel insights into the structural basis of iron donation for FeS cluster biogenesis and novel FeS repair proteins have been identified. This increased our understanding of the processes that involve FeS cluster containing proteins and advanced the research that focuses on these systems.
The results obtained, have been disseminated at international events and meetings: around 80 participations of TIMB3 members at events across the globe, including Europe, USA, Canada and China, which took place in person or in virtual/hybrid modes.
Taken together, the achievements related to a number of structurally and functionally distinct metalloproteins, side by side with development of the novel spectroscopic methodologies for their studies, are expected to provide progress beyond the state of the art in several fundamental and applied scientific areas. These include medicine, biomedicine, biotechnology, pharmaceutical, environmental, textile, paper, food and chemical industries, as well as biological hydrogen production and hydrogenase-based biofuel cells development fields, among others, thus ensuring a wide socio-economic impact.