Evolutionary changes in key protein synthesis enzyme, isoleucyl-tRNA synthetase (IleRS), may help bacteria fine-tune how quickly proteins are made, report researchers from Institute of Science Tokyo, Japan; the University of Zagreb Faculty of Science, Croatia; and ETH Zürich, Switzerland. Researchers traced the enzyme's evolutionary history and identified three distinct IleRS clades. Their findings show that C-terminal remodeling, including repeated loss of a structural region called "INS," may have helped refine enzyme activity and reaction speed.
Every cell contains the millions of protein molecules necessary for various cellular functions. Protein synthesis is the process through which the information encoded in our DNA is converted into the proteins needed for sustaining life. During this process, transfer RNAs (tRNAs) act as molecular carriers, bringing specific amino acids to the ribosome, where they are assembled into proteins according to the genetic information encoded in messenger RNA (mRNA), which is derived from DNA. Before a tRNA can perform this role, it must be "charged" with the correct amino acid. This crucial step is carried out by a family of enzymes called aminoacyl-tRNA synthetases (AARSs), which recognize specific tRNAs and then attach the corresponding amino acid to the enzyme (aminoacylation). The accuracy of this process is essential because attaching the wrong amino acid can lead to errors in the synthesized protein.
One member of this family is isoleucyl-tRNA synthetase (IleRS), which pairs the amino acid isoleucine to its corresponding tRNA. IleRS has traditionally been classified into two forms, IleRS1 and IleRS2, based on differences in their C-terminal regions. However, the evolutionary history and functional significance of these differences have remained unclear.
Now, a study led by Professors Liam M. Longo from the Earth-Life Science Institute, Institute of Science Tokyo, Japan; Ita Gruic-Sovulj from the Department of Chemistry, University of Zagreb Faculty of Science, Croatia; and Nenad Ban from ETH Zürich, Switzerland, reveals a more complex picture of how this essential enzyme has evolved. The study combines bioinformatics, X-ray crystallography, and extensive kinetic characterization, reconstructing the evolutionary history of IleRS and investigating how evolutionary changes in the enzyme affect tRNA recognition, enzyme function, and aminoacylation rate.
The study was published in the journal Nature Communications on October 12, 2026.
"Our findings suggest that bacteria have repeatedly streamlined parts of this enzyme, potentially enhancing aminoacylation rate and contributing to faster protein synthesis," notes Longo.
To uncover the evolutionary details, the researchers analyzed IleRS sequences from bacteria, archaea, and eukaryotes and constructed phylogenetic trees for several of the enzyme's domains. Their analysis revealed three evolutionary groups: IleRS1, IleRS2, and a previously unrecognized group of deep-branching archaeal enzymes, which they named IleRS-A. The researchers found that IleRS-A has a C-terminal organization resembling IleRS2, while its synthetic domain (referred to as HUP) is more closely related to that of IleRS1. Moreover, their analysis suggests that IleRS1 could have emerged from an IleRS-A-like ancestor, accompanied by remodeling of its C-terminal region.
To further support this evolutionary model, the researchers also found evidence that C-terminal remodeling has continued throughout IleRS evolution. In particular, an approximately 80-amino-acid region called the INS subdomain has been independently lost in multiple prokaryotic lineages. The INS subdomain interacts with the D-loop (a part of tRNA) in IleRS2. To investigate its functional significance, the team determined the crystal structure of IleRS2 from Priestia megaterium bound to a tRNA from Escherichia coli. They analyzed the structural model using targeted mutations and kinetic experiments. This was the first reported crystal structure of an IleRS containing the evolutionarily unstable INS subdomain.
The experiments revealed that while the INS subdomain does contribute to tRNA binding, it is not essential for productive aminoacyl transfer under the conditions assayed. Mutations that weakened the interaction between INS and the tRNA D-loop increased the enzyme's catalytic turnover by about fourfold. Removing the entire INS subdomain reduced the enzyme's apparent affinity for tRNA, increasing the KM for tRNA by approximately 15-fold but did not abolish aminoacylation activity—demonstrating how readily loss of the INS domain can be accommodated.
The researchers also tested these mutants in E. coli. When the bacteria's own IleRS was blocked, the modified enzymes carrying mutations in the INS subdomain were still able to support bacterial growth. Although complete removal of INS made complementation more difficult, the mutant INS enzyme remained functional when expressed at higher levels together with additional tRNA for isoleucine. In contrast, mutations that disrupted the tRNA anticodon recognition site prevented the enzyme from supporting bacterial growth, highlighting the critical role of anticodon recognition in aminoacyl transfer.
"We observed that evolutionary changes to the C-terminal region can alter how IleRS interacts with tRNA without necessarily disrupting its essential catalytic function," says Gruic-Sovulj.
The findings showcase the C-terminal region of IleRS as an important site of evolutionary remodeling through which the enzyme's interactions with tRNA and catalytic activity can be tuned. The authors suggest that C-terminal remodeling, including the replacement of a domain during the emergence of IleRS1 and the repeated loss of INS in prokaryotes, may have contributed to faster aminoacylation. Overall, the study shows how an essential component of the translation machinery can maintain its function while undergoing structural changes that may influence the speed of its catalytic cycle.
