Abstract EANA2026-39

Understanding how amino acids were incorporated into the protein synthesis system is important for investigating the origin of life and early molecular evolution. Although modern protein synthesis utilizes 20 amino acids, earlier systems likely employed a more limited set, with additional amino acids gradually added during evolution. However, the detailed process underlying this incorporation remains unresolved.

In the present study, we focused on histidine, whose imidazole side chain is involved in proton transfer and metal-ion coordination, making it important for diverse enzymatic reactions. Because of its essential metabolic roles, a stable supply of histidine would likely have been required before histidine could be incorporated into the translation system. Achieving such a stable supply likely required the emergence of an independent biosynthetic pathway rather than relying on environmental sources. Yet, modern histidine biosynthesis depends on enzymes that themselves contain histidine residues. This creates a potential evolutionary “chicken-and-egg” paradox: histidine biosynthesis requires proteins that already rely on histidine for their function. We hypothesized that primitive histidine-free enzymes existed during the early evolution of this pathway and aimed to experimentally reconstruct such ancestral enzyme states.

As an initial step toward reconstructing histidine-free enzymes, we analyzed sequence conservation of each enzyme in the histidine biosynthetic pathway (HisA–HisN), with particular attention to conserved histidine residues in their active sites.We found that active-site histidine residues are not conserved across species in several enzymes. In contrast, highly conserved histidine residues were identified in HisA, HisB, HisD, and HisH. Notably, HisB and HisD contained five and three highly conserved histidine residues, respectively, with histidine retained in more than 98% of the analyzed sequences. 

We therefore focused on HisD, which catalyzes the final step of histidine biosynthesis. Detailed analysis of conserved active-site residues revealed alternative amino acids at these conserved positions in several species within the phylum Cyanobacteriota, including Acaryochloris, Gloeothece, Microcystis, Synechocystis, and Hydrococcus. In these species, histidine residues are found at different positions within the protein. Interestingly, these positions vary among species, suggesting multiple evolutionary strategies in the positioning and functional roles of histidine residues. 

Among these cyanobacterial species, we focused on Hydrococcus sp. Prado102, whose HisD protein contains relatively few histidine residues overall. We then designed and constructed chimeric genes by exchanging the regions surrounding the conserved histidine residues between E. coli and Hydrococcus proteins. The resulting chimeric proteins, together with wild-type proteins from both organisms, were expressed and purified. Enzymatic assays were established using the E. coli wild-type enzyme. Functional characterization of the mutant enzymes and Hydrococcus-derived proteins is currently in progress. These analyses are expected to illuminate how histidine was recruited as a catalytic residue in its own biosynthetic pathway during early molecular evolution.