Following the first proteins

Weizmann Institute of Science scientists created a prototype of an ancient enzyme in the laboratory. Their findings indicate the possibility that the first proteins were a kind of "helpers" for DNA and RNA

Even the simplest bacterium has an arsenal of sophisticated enzymes - active multi-site proteins that carry out the basic tasks of life. The complexity of enzymes has ancient roots, but how were the first proteins formed? Weizmann Institute of Science scientists and their research partners recently offered a possible answer to this question with the help of a protein they created in the laboratory. Their findings, published in the "Records of the American Academy of Sciences" (PNAS), may shed new light on the earliest stages in the history of the living cell - about 3.7 billion years ago.

From bacteria to humans, phosphate-binding loops (P-loops for short) are included in various enzymes in all living things. Based on previous studies and many years of experience, the research team, led by Dr. Maria Luisa Romero-Romero from Prof. Dan Toufik's group in the Department of Biomolecular Sciences at the institute, marked these loops as a possible starting point for the production of a prototype of the ancient enzyme. These short loops, About eight amino acids in length, cooperate with another molecule found in every living being: ATP (adenosine triphosphate).

The energy economy of the animal world uses ATP - a molecule even more ancient than proteins - as a kind of "trading currency" with which it is possible to "translate" and convert different forms of energy into each other, according to the unique needs of each living system. Enzymes pick up a phosphate group from the ATP molecule and thus use it in various biochemical reactions they carry out. These reactions include the attachment of phosphate groups to other metabolites, for example sugars, nucleic acids or amino acids, which are activated in this way - similar to inserting a coin into a slot machine. Thus, the phosphorylation-binding loops, which carry and release the phosphorylated groups, are essential for the functioning of almost all living cell systems.

Our proteins seem to us to be a finished work, and we find it difficult to imagine the 'carts' and 'wheelbarrows' that preceded them. We believe that our research shows a protein that is equivalent to one of those wheelbarrows"

On the basis of protein sequences based on the Zarha loop that exist today in nature, the scientists identified the earliest sequence of this loop and designed new protein sequences that would contain the same sequence. The criteria set for the proteins were: that they be as short as possible and allow the activity of the phosphorylation loop - but no more. Prof. Toufik says: "The fact that these small proteins worked well helps us understand how the first enzymes developed from short sequences, which at a certain point began to bind to each other and accumulate more and more functions." The research group also included the laboratory team of Prof. Michal Sharon from the Institute and the teams of Professors Gabriel Varani and David Baker from the University of Washington and researchers from Germany, Singapore and Norway.

The surprise was that the phosphorylation loops in the designed protoenzymes not only bound ATP, but also bound tightly to DNA and RNA - an activity that is not characteristic of today's phosphorylation loop proteins. This discovery may shed new light on the scenario according to which the origin of life is in DNA and RNA molecules. According to this scenario, which is now increasingly accepted in the scientific community, RNA molecules were responsible for the actions currently reserved for enzymes. Says Prof. Toufik: "Our findings point to the possibility that the first proteins were a kind of 'helpers' for DNA and RNA. They were probably multi-purpose and relatively simple, and with the progress of evolution they developed into enzymes with increasing efficiency and specificity.

"In this study, we showed how a complex mechanism actually develops from simpler parts - even when it seems that it is too complex to allow decomposition into elementary elements. Our proteins seem to us to be a finished work, and we find it difficult to imagine the 'carts' and 'wheelbarrows' that preceded them. We believe that the research Ours presents a protein that is the equivalent of one of those wheelbarrows, or in Darwin's words: '[Life] has evolved from such a simple beginning into infinite beautiful forms without A limit, limitless wonders, which are still being added and developed.''

The protein sequences produced in the laboratory were extremely short, some of them only 55 amino acids long - about 1/6 of the length of an average enzyme.

The proteins of the future - the green route
If the above study followed the past of the proteins, another study published recently may indicate the proteins of the future. A research team with the participation of Prof. Dan Toufik and his group succeeded in creating a new enzymatic pathway, which may help plants make more efficient use of carbon dioxide (CO2), thus increasing agricultural produce and enabling a better deal with future food shortages.

Plants absorb CO2 from the air and convert it, through photosynthesis, into carbon-based proteins and sugars (carbon fixation process). In doing so, they not only provide food and breathing oxygen for the rest of life on our planet, but also keep CO2 levels naturally low. However, in a side reaction that damages the plant, the rubisco enzyme binds to the oxygen released in photosynthesis and produces a compound known as glycolate. To prevent its accumulation, there is a secondary enzymatic pathway in the plant which breaks down the glycolate. Unfortunately, this process uses quite a bit of the plant's energy and may also result in the release of CO2 back into the atmosphere. The scientists decided to engineer a new enzymatic pathway: instead of breaking down the glycolate, the compound is recycled and the carbon in it is translated into sugars instead of being released into the air as CO2. Dr. Arne Bar-Aven from the Max Planck Institute for Plant Molecular Physiology in Potsdam-Golem, Germany, and Dr. Sheral Fleischman from the Institute's Department of Biomolecular Sciences participated in the study.

The planned route does not exist in nature, and it was designed from start to finish by the research team. In contrast to the natural breakdown pathway of glycolate, in which more than ten enzymes are involved, the research team created two new enzymes that work alongside two natural enzymes in the cycle of glycolate into reusable components. As predicted by the computer models, the route worked in a laboratory experiment in a process that was efficient - using about half of the energy the plant needs to get rid of glycolate and without losing CO2. The researchers hope that they will be able to introduce the glycolate cycle pathway into photosynthetic bacteria or plants.

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