Substrate Affinity Versus Catalytic Efficiency: Ancestral Sequence Reconstruction of tRNA Nucleotidyltransferases Solves an Enzyme Puzzle

In tRNA maturation, CCA-addition by tRNA nucleotidyltransferase is a unique and highly accurate reaction. While the mechanism of nucleotide selection and polymerization is well understood, it remains a mystery why bacterial and eukaryotic enzymes exhibit an unexpected and surprisingly low tRNA substrate affinity while they efficiently catalyze the CCA-addition. To get insights into the evolution of this high-fidelity RNA synthesis, the reconstruction and characterization of ancestral enzymes is a versatile tool. Here, we investigate a reconstructed candidate of a 2 billion years old CCA-adding enzyme from Gammaproteobacteria and compare it to the corresponding modern enzyme of Escherichia coli. We show that the ancestral candidate catalyzes an error-free CCA-addition, but has a much higher tRNA affinity compared with the extant enzyme. The consequence of this increased substrate binding is an enhanced reverse reaction, where the enzyme removes the CCA end from the mature tRNA. As a result, the ancestral candidate exhibits a lower catalytic efficiency in vitro as well as in vivo. Furthermore, the efficient tRNA interaction leads to a processive polymerization, while the extant enzyme catalyzes nucleotide addition in a distributive way. Thus, the modern enzymes increased their polymerization efficiency by lowering the binding affinity to tRNA, so that CCA synthesis is efficiently promoted due to a reduced reverse reaction. Hence, the puzzling and at a first glance contradicting and detrimental weak substrate interaction represents a distinct activity enhancement in the evolution of CCA-adding enzymes.

Automated summary

CCA addition is a unique and highly accurate reaction in tRNA maturation. While the mechanism is well-known, it is still a mystery why bacterial and eukaryotic enzymes exhibit low tRNA substrate affinity but efficiently catalyze the reaction. Through the reconstruction and characterization of an ancestral enzyme from Gammaproteobacteria, researchers discovered that the ancestral enzyme has higher tRNA affinity and lower catalytic efficiency compared to the modern enzyme from Escherichia coli. They also found that the modern enzyme increased polymerization efficiency by reducing binding affinity to tRNA. This puzzling weak substrate interaction represents a distinct activity enhancement in the evolution of CCA-adding enzymes.