Protein-protein interactions in prokaryotic heme biosynthesis: Substrate channeling, electron transfer and possibly more

Background.

Several enzymatic steps are required to build heme, and each biosynthetic step is catalyzed by a specific enzyme (Dailey et al., 2017). If we focus only on the last four reactions of the coproporphyrin-dependent heme biosynthetic pathway, which is almost unique to Gram-positive bacteria (including many problematic multidrug-resistant pathogens), we have a rather diverse group of enzymes to study. These enzymes are of different subunit structure, oligomeric state (monomer to pentamer), and utilize a variety of co-factors for catalysis (e. g. [2Fe-2S] cluster, FAD, iron porphyrin) (Falb et al., 2023).
Nevertheless, every product of an enzyme is the substrate of another enzyme. Let alone that these substrates are either strong chelators or highly potent redox molecules that should not accumulate in a non- compartmentalized Gram-positive cell. Therefore, a strict substrate/product channeling mechanism is proposed, which must be facilitated by the enzymes involved through protein-protein interactions (PPI). In addition, still unknown heme b (final product) accepting proteins must interact with the final enzyme (coproheme decarboxylase) of this biosynthetic pathway.

Aims and Methods.

In this project we aim to investigate PPI within the coproporphyrin-dependent heme biosynthesis pathway in a holistic approach. On the one hand we will use a bacterial adenylate cyclase-based two hybrid system (BACTH) and a library approach to identify yet unknown protein interaction partners using a specific model organism (e.g. Bacillus subtilis) (Karimova et al., 2024). Furthermore, we will establish a high-throughput cell-based fluorescent Nanobit assay, to analyze general PPI. We will follow up on positive hits on a purified protein level, performing in-depth analysis of PPI using high-end biophysical methods (e.g., surface plasmon resonance, isothermal titration calorimetry, multi-angle laser light scattering, X-ray crystallography, and others). Identified protein interaction partners will be analyzed by computational methods to further rationalize and understand the driving forces of interaction.
The coproporphyrin-dependent heme biosynthesis pathway differs significantly to the protoporphyrin-dependent heme biosynthesis pathway present in human and Gram-negative bacteria. This makes every enzyme of this pathway and their specific interactions prominent targets for steric or mechanistic inhibition. These inhibition strategies serve as a starting point for the development of novel antibacterial agents, which are urgently needed in view of the high number of Gram-positive pathogens that are resistant to many common antibiotics.

REFERENCES
1. Dailey, H.A., Dailey, T.A., Gerdes, S., Jahn, D., Jahn, M., O'brian, M.R., and Warren, M.J. (2017). Prokaryotic Heme Biosynthesis: Multiple Pathways to a Common Essential Product. Microbiol Mol Biol Rev 81.
2. Falb, N., Patil, G., Furtmüller, P.G., Gabler, T., and Hofbauer, S. (2023). Structural aspects of enzymes involved in prokaryotic Gram-positive heme biosynthesis. Comput Struct Biotechnol J 21, 3933-3945.
3. Karimova, G., Gauliard, E., Davi, M., Ouellette, S.P., and Ladant, D. (2024). Protein-Protein Interaction: Bacterial Two Hybrid. Methods Mol Biol 2715, 207-224.