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Reconstruction of a two-billion-year-old enzyme solves a long-standing mystery

Reconstruction of a two-billion-year-old enzyme solves a long-standing mystery

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Biotechnology Bioinformatics DNA

The research team reconstructed an ancestral enzyme by searching databases for corresponding modern enzymes, used the obtained sequences to calculate the original sequence, and introduced the corresponding gene sequence into laboratory bacteria to produce the desired protein. The enzyme was then studied in detail and compared to modern enzymes.

Molecular biologists and bioinformaticians did detective work to accomplish this feat.

The research team, led by Professors Mario Mörl and Sonja Prohaska, focused on enzymes called tRNA nucleotidyltransferases, which attach three nucleotide building blocks in the sequence CCA to small RNAs (transfer RNAs) in cells. These RNAs are then used to provide amino acids for protein synthesis. Using phylogenetic reconstructions, the team reconstructed a candidate ancestral enzyme that existed in bacteria around 2 billion years ago and compared it to a modern bacterial enzyme.

They found that both enzymes work with similar precision, but differ significantly in their reactions. Until now, scientists have not been able to understand why modern enzymes often pause their activity, but this study showed that this tendency is actually an evolutionary advantage that puzzled biochemists for decades.

The ancestral enzyme is processive, ie it works without interruption, but occasionally removes nucleotide building blocks that have already been correctly attached. The results show that one can learn a lot about the evolution and properties of modern enzymes from enzyme reconstructions and that many questions can only be solved by the interaction of bioinformatics and biochemistry – in a back and forth between computer calculations and laboratory experiments.

Two billion year old enzyme reconstructed

This is what a family tree looks like whose origin (middle) goes back two billion years. The tips of the branches each represent the enzyme of a modern organism. Credit: Diana Smikalla

Float into the past by tracking down relationships

Gene sequences can also be used to create evolutionary family trees of bacteria. Based on today’s great diversity of organisms in a species tree, the evolutionary path of individual genes can be reconstructed along relationships and branches and meticulously traced back to a common origin.

The reconstruction is essentially a three-step process. First, databases are searched for corresponding modern enzymes in order to be able to examine the sequence of amino acid building blocks. The sequences obtained can then be used to calculate what the original sequence should have looked like. The corresponding gene sequence, which codes for the old enzyme, is then introduced into laboratory bacteria so that they produce the desired protein. The enzyme can then be examined in detail for its properties and compared with modern enzymes. “When the news came back from the laboratory that the reconstructed enzyme carried out the CCA addition, and that even in a wider temperature range than today’s enzymes, that was the breakthrough,” remembers Sonja Prohaska.

Evolutionary Optimization: Activity breaks increase efficiency

Like organisms, enzymes are also optimized through evolution. The work (catalysis) of an enzyme usually runs faster and better, the stronger it can bind its substrate. The reconstructed ancestral enzyme does just that, holding on to the substrate, the tRNA, and attaching the three CCA nucleotides one by one without releasing them. Modern tRNA nucleotidyl transferases, on the other hand, are distributive, ie they work step by step with pauses in which they release their substrate again and again. Nevertheless, they are more efficient and faster than their traditional predecessors. This confused the researchers. Why do modern enzymes keep releasing their substrate? The explanation lies in the phenomenon of the reverse reaction, in which the built-in nucleotides are removed again by the enzyme. While the tight binding of the ancestral enzyme to the substrate leads to subsequent removal, the release of the substrate in modern enzymes almost completely prevents the reverse reaction. This allows them to work more efficiently than their predecessors.

“We were finally able to explain why modern tRNA nucleotidyl transferases work so efficiently despite their distributive character,” says Mario Mörl. “The finding completely surprised us in the team. We didn’t expect anything like this. We already had the question 20 years ago and can now finally answer it with bioinformatic reconstruction methods. This close cooperation between bioinformatics and biochemistry has existed in Leipzig for several years and, not for the first time, has proven to be a great advantage for both sides”.

Reference: “Substrate Affinity Versus Catalytic Efficiency: Ancestral Sequence Reconstruction of tRNA Nucleotidyltransferases Solves an Enzyme Puzzle” by Martina Hager, Marie-Theres Pöhler, Franziska Reinhardt, Karolin Wellner, Jessica Hübner, Heike Betat, Sonja Prohaska and Mario Mörl, November 21 2022, Molecular Biology and Evolution.
DOI: 10.1093/molbev/msac250

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