Compartmentalization of biosynthetic enzymes in bacterial cells: the histidine metabolic pathway case

Introduction: It is known that the inner concentration of proteins within the cell cytoplasm is so high that limits the diffusion of enzymes and metabolic intermediates, leading to a loss of time and energy. Therefore, the organization of genes in operons would have enabled to have enzymes involved in the same metabolic pathway physically close to each other. A corollary to this hypothesis in the possibility of physical interactions between the enzymes of the same metabolic pathway, resulting in the formation of a supramolecular complex capable in channeling the intermediates from one enzyme to a physical adjacent one, with restricted diffusion in the surrounding milieu. Objectives: The aim of this study is to try to demonstrate the existence of the compartmentalization of proteins involved in the same metabolic pathway, and the formation of multi-enzymatic complexes in bacterial cells, using as model system the histidine biosynthesis. In Escherichia colithe genes involved in this metabolic pathway are organized in a compact operon. At least 7 of the 10 genes of the pathway underwent different gene fusions in different evolutionary lineages, suggesting a compartmentalization of the biosynthetic enzymes. Materials & Methods: To evaluate the possible interactions between the enzymes of this pathway a combination of different approaches has been used: i) the Bacterial Adenylate Cyclase Two-Hybrid (BACTH) system, ii) the β-galactosidase assay, iii) the His-tag method, and iv) microscopy analyses. Results: So far, using the BACTH system, the interaction between the enzymes His-H and His-F has been validate, as a positive control of the system. Conclusion: The testing of all the other possible interactions between the enzymes of the histidine biosynthetic pathway, through the proposed multidisciplinary approach, is in progress to evaluate if they form a single big multi-enzymatic complex.