Biochemical and functional characterization of proteins and enzymes for heme-iron acquisition and utilisation in bacteria

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Characterization of proteins of poorly understood pathways from microorganisms is important to increase our knowledge of biological processes, to discover new protein functions and to identify new protein targets, which are highly desirable to help the development of much needed new antimicrobial molecules. The long-term objective of my research program is to decipher the structure-function relationships of proteins and enzymes of key bacterial processes, such as that of iron and heme acquisition and metabolism, in order to increase knowledge and to provide new targets and approaches to fight bacterial infections. We aim to characterize an 8-gene cluster involved in heme acquisition and utilisation from the enterobacterium Escherichia coli O157:H7 that causes food-borne infections in humans. We characterized in the past years 3 of the cytoplasmic Chu proteins (ChuS, ChuX and ChuY) and we propose further studies as their individual function is still not established and the biology of the Chu pathway is poorly understood. Our work shows that ChuS is an enzyme that degrades heme using molecular oxygen and hydrogen peroxide as the oxidants and producing a verdoheme intermediate, which is the same intermediate produced by the well-known heme oxygenase. However, the next step, the opening of the porphyrin, is catalyzed by a novel mechanism that generates a tripyrrole porphyrin product, hematinic acid, carbon monoxide and free iron. This novel mechanism of opening of the porphyrin from the verdoheme will be investigated using kinetic and spectroscopic approaches. To validate that this reaction detected in a test tube also occurs in vivo, we aim to identify the porphyrin degradation products from the native bacterium using a targeted metabolomics approach. The function(s) of the Chu proteins will be investigated in a non-infectious physiological model. Finally, we propose to probe in vitro and in vivo protein-protein complexes predicted among the cytoplasmic Chu proteins to understand their importance for catalysis. The proposed research will increase our fundamental knowledge of key enzymes involved in heme-iron acquisition in enterobacteria, and in other bacteria that possess such pathway. A major strength of our proposal is that the investigation of the activities and mechanisms of Chu proteins will be performed both in the test tube and in the bacterial cells. Hence, our results will provide a solid, reliable basis to evaluate one or more of these proteins as potential novel target(s) for the development of antimicrobial strategies. On the longer term, our studies may have a significant impact for the development of new antimicrobials, thus to be important to human health and to economic sectors such as agriculture (animal health). This research will also contribute to the training of highly qualified personnel in the dynamic areas of protein science, biochemistry and physiology who are in high demand in academia and biotechnology industries.