Abstract EANA2026-72

The stability and evolution of functional biomolecules are important for understanding the history of life on Earth and for considering how such molecules may behave in broader planetary environments. However, the stability of ancestral enzymes in microbial lineages with limited ecological and evolutionary information remains unclear. Candidate phyla radiation (CPR) bacteria are characterized by reduced genomes, limited metabolic capacities, few cultured representatives, and unresolved evolutionary placement. These features make CPR bacteria a useful target for examining molecular properties in microbial lineages whose ecology, habitats, and ancestral characteristics are still poorly understood. Ancestral sequence reconstruction (ASR) enables experimental analysis of inferred ancestral proteins and is therefore useful for investigating protein properties that cannot be observed directly. In this study, we estimated ancestral CPR lysyl-tRNA synthetase (LysRS) sequences and measured the thermostability of reconstructed proteins. LysRS genes are conserved among many CPR phyla, making this enzyme suitable for ancestral reconstruction. In the LysRS phylogenetic trees used for ASR, CPR and non-CPR LysRS sequences diverged at the deepest bacterial node. Because ASR inherently involves uncertainty, multiple ancestral CPR LysRS sequences were reconstructed using different phylogenetic tree topologies and ancestral sequence inference programs. The reconstructed ancestral CPR LysRS proteins retained high thermostability. The LysRS phylogenies inferred in this study differed from currently proposed organismal phylogenies and therefore primarily reflected the evolutionary history of LysRS itself. Our results show that ASR can experimentally estimate the thermostability of ancestral enzymes even in bacterial lineages with limited information about their ancestral characteristics and habitats. These data may provide clues for considering enzyme thermostability in ancient environments on early Earth and in potentially habitable planetary environments, where direct molecular evidence is extremely limited.