An enzyme from the bacterium Priestia megaterium offered researchers a close look at how tiny structural changes could speed a key step in making proteins. In a study published in Nature Communications, scientists found that weakening one part of the enzyme’s grip on a carrier molecule increased its catalytic turnover about fourfold.
Cells need proteins to carry out many jobs, and making them depends on matching each amino acid to the correct transfer RNA, or tRNA. These small molecules carry amino acids to the cell’s protein-making machinery. The enzyme isoleucyl-tRNA synthetase, known as IleRS, attaches the amino acid isoleucine to its matching tRNA. If this step goes wrong, the wrong building block can enter a protein.
Researchers from the Institute of Science Tokyo in Japan, the University of Zagreb Faculty of Science in Croatia, and ETH Zürich in Switzerland traced how IleRS has changed over evolutionary time. The team, led by Liam M. Longo, Ita Gruic-Sovulj and Nenad Ban, combined computer analysis of enzyme sequences with X-ray crystallography, which reveals molecular structures, and tests of enzyme activity.
Their analysis identified three evolutionary groups: IleRS1, IleRS2 and a newly recognized group of deep-branching archaeal enzymes, named IleRS-A. Archaea are single-celled organisms distinct from bacteria and eukaryotes, the group that includes animals, plants and fungi. The researchers found that IleRS-A has a C-terminal region resembling IleRS2, while another part, the synthetic domain, is more closely related to IleRS1. Their findings suggest that IleRS1 may have descended from an ancestor resembling IleRS-A, with changes to its C-terminal region along the way.
One change appeared more than once. An approximately 80-amino-acid segment called the INS subdomain was independently lost in several groups of prokaryotes, a broad category that includes bacteria and archaea. In IleRS2, INS contacts the D-loop, a region of tRNA. To examine what that contact does, the team determined the first reported crystal structure of an IleRS containing this evolutionarily unstable segment. The structure showed IleRS2 from Priestia megaterium bound to a tRNA from Escherichia coli.
Experiments showed that INS helps the enzyme bind tRNA, but was not essential for transferring the amino acid under the conditions tested. Mutations that weakened the contact between INS and the tRNA’s D-loop raised catalytic turnover about fourfold. This suggests that a looser interaction can let the enzyme complete its work faster, although the study does not establish how much this changes protein production inside living cells.
The results offer a clearer picture of how a basic step in protein making may have been tuned during evolution. The researchers say repeated streamlining of IleRS in bacteria could have improved aminoacylation, the attachment of an amino acid to tRNA, and contributed to faster protein synthesis. Further work can test how these enzyme changes affect cells in their natural settings.