Dissecting the metabolic plasticity of Actinobacteria
Final Report Abstract
The main objective of the oberall German-French project was to shed more light on selected biochemical reactions located at two key nodes of central metabolism in Actinobacteria, i.e., their pyruvate and the 2-oxoglutarate node, which are relevant both for pathogenic and biotechnologically relevant species. The experimental approaches employed during the project include bacterial genetics, such as the construction of a large number of plasmids and strains, protein biochemistry, in particular purification and characterization of enzymes, and structural biology by crystallography, cryo-electron microscopy, and modelling. The main results of our part of the overall project (WP 2 and 3.3) can be resumed as follows: We studied the function and regulation of the pyruvate:quinone oxidoreductase (PQO), which oxidizes pyruvate to acetate and CO2 with menaquinone as electron acceptor and thus provides an alternative route of pyruvate oxidation besides the PDH complex. The results show that the activity is severely influenced by the carbon source and the growth phase and hint at a regulation of PQO by posttranslational modification, i.e. by acylation of specific lysine residues, which all are located at the surface of PQO and when substituted by other amino acids, lead to altered PQO activities. It was shown that PQO activity in C. glutamicum decreased gradually with increasing external pH and with increasing growth rate. The PQO enzyme was purified to homogeneity and biochemically characterized. Co-culture experiments with C. glutamicum WT and the PQO-deficient mutant strain showed the same growth behaviour under all conditions tested, indicating that active PQO does not confer a significant advantage or disadvantage to WT cells. The crystal structure of PQO was solved with a high resolution. The enzyme is a homotetramer that shares similarity with the E. coli pyruvate oxidase PoxB. Regions within the active site of PQO have been identified that interact with and stabilize the cofactors TPP and FAD, and may play critical roles in the catalytic process. Despite substantial similarities between PQO and PoxB, structural differences were observed, specifically in the C-terminal region. The structural and biochemical characterization of C. glutamicum PQO suggests regulation mechanisms that involve protein-membrane interactions as well as specific post-translational modifications. The C-terminal 17 amino acids in the PQO enzyme, which include the amphipathic α-helix, are crucial for the enzyme's stability, detergent/lipid binding, and potentially its enzymatic activation.
