Advanced spectroscopic study aiming to the understanding of the heme biosynthetic pathway of Gram-positive bacteria

Heme b is an iron porphyrin that acts as a prosthetic group for many enzymes involved in various physiological processes. Bacterial pathogens need this porphyrin to survive and infect the host. In 2015, it was discovered that Gram-positive bacteria and humans produce heme b via different pathways, using different enzymes, substrates and products. Gram-positive bacteria rely on coproporphyrin III (cpIII) in the coproporphyrin dependent (CPD) pathway, while humans use protoporphyrin IX (ppIX) in the protoporphyrin dependent (PPD) pathway. CpIII contains four propionate groups in positions 2, 4, 6 and 7, while ppIX presents two vinyl groups in positions 2 and 4. Before the discovery of the CPD pathway, the Gram-positive bacteria biosynthetic pathway was investigated using either the ppIX or other two-vinyl porphyrins as substrate. This gave rise to confusing results. Due to the increasing antibiotic resistance in many bacteria, a comprehensive understanding of the enzymes involved in the CPD pathway, using the physiological substrates, is essential for the development of therapeutic drugs targeting this biosynthetic mechanism. My PhD research was focused on the study of the heme b synthesis by antibiotic resistant Gram-positive bacteria. Using UV-vis electronic absorption and resonance Raman (RR) spectroscopies on the enzymes involved in the CPD pathway at different temperatures (80 and 298 K), pH values, and in the presence of exogenous ligands, I was able to provide the first systematic study of the bacterial biosynthesis using the physiological substrates. Furthermore, during my stay at Prof. Hofbauer’s laboratory (BOKU University, Wien, Austria) I have expressed and purified site-directed variants, and performed ligand binding characterization via steady-state and pre-steady-state kinetics.