Biochemistry of HemQ

Recently, a novel heme b biosynthesis pathway has been detected in Gram-positive bacteria with the last step being catalysed by HemQ, an enzyme that is not found in human metabolism. Despite of its importance there is only little knowledge about its biochemistry and the mechanism of its action is completely unknown. Thus this basic research project focuses on the elucidation of structure-function relationships of HemQ. By applying a broad set of biochemical and biophysical techniques this interdisciplinary project aims at investigating four model proteins from different phylogenetic lineages in order to establish a general reaction mechanism. In detail we have selected three HemQs from important human pathogens, namely Listeria monocytogenes, Staphylococcus aureus and Corynebacterium diphteriae, thus including dangerous (antibiotics-resistant) and wide- spread hospital bugs. Additionally, HemQ from the Archaeum Sulfolobus sulfataricus will be investigated. The model proteins will be produced recombinantly, mutated and analysed in a comprehensiveand comparativebiochemicalstudy including biospectroscopies, kinetic, thermodynamic and redox studies, X-ray crystallography and computational methods. Based on the obtained knowledge of action of HemQ at a molecular basis, it will be possible to rationally design inhibitors against HemQ in the future. Consequently, this study will later on allow to create a new class of antibiotics against these wide-spread pathogens. These bacteria are strongly dependent on the function of many heme-containing enzymes including those that are directly involved in host attack. Thus inhibition of heme biosynthesis will immediately block the viability of these pathogens.

In this project, the reaction and action mechanism of the enzyme coproheme decarboxylase was investigated in detail and largely succesfully elucidated. This enzyme catalyzes the final step of heme biosynthesis in very many, mainly Gram-positive, bacteria. Among them are some dangerous pathogens that exhibit multiple resistances to common established antibiotics. These resistant pathogenic bacteria are dangerous hospital germs responsible for a large number of deaths annually worldwide. Coproheme decarboxylase is a promising target for the development of novel antibiotically active compounds, as no structurally related protein is found in humans and inhibition of this enzyme is lethal to the pathogen. In order to target the search for suitable inhibitors, it is of great importance to elucidate the basic structure-function relationships of this protein. This project is a classical basic research project in which we have managed to reliably and reproducibly produce and purify the target protein and introduce targeted point mutations. The comparative biochemical and biophysical studies of the wild- type protein and the numerous protein variants allowed us to explore the individual steps of the multi-step and complex redox reaction. This knowledge is the basis for the development of highly specific inhibitors that aim to mechanistically influence the reaction by manipulating the electron fluxes during the redox reaction. A particular research success, in addition to elucidating the redox chemistry, was also being able to observe the reorientation of the substrate during the reaction. These structural biological findings are the perfect basis for the quest for inhibitor candidates that induce a steric blockade that makes it impossible for the enzyme to process the substrate correctly. These results were published in well-known and renowned scientific journals in several articles. The knowledge gained in the course of this project has led to further important and interesting questions, which will enable further targeted and promising research.