Study explored how restricting the amino acid diversity in ancestral enzymes can uncover alternative catalytic functions, including ATP formation from ADP

Researchers in Japan engineered ancestral nucleoside diphosphate kinase variants with restricted amino acid repertoires to explore alternative catalytic functions. One variant, Arc1-12KR, retained its protein fold despite lacking catalytic histidine and unexpectedly converted two ADP molecules into ATP and AMP. The findings reveal alternative catalytic potential in simplified proteins and offer clues to possible routes of energy metabolism during early protein evolution.

Proteins perform essential functions in living organisms, from catalyzing biochemical reactions to supporting cellular energy metabolism. Their functions depend on their amino acid composition and three-dimensional structures. However, early proteins may have evolved when fewer amino acid types were available than those used by most modern proteins. Understanding how proteins built from a more limited set of amino acids could perform essential biochemical reactions may therefore offer insights into the origins and evolution of life.

To investigate this possibility, Professor Satoshi Akanuma and Assistant Professor Sota Yagi of Waseda University, Japan, along with Subrata Dasgupta and Shunsuke Tagami of the RIKEN Center for Integrative Medical Sciences, Japan, examined how restricting amino acid diversity affects the structure and function of ancestral enzymes. Explaining the motivation behind the study, Akanuma said, “We were interested in what happens when an ancestral enzyme is rebuilt using a reduced set of amino acids that may resemble the chemical limitations of early life.” The study was published online in Angewandte Chemie International Edition on October 09, 2026.

The researchers engineered variants of ancestral nucleoside diphosphate kinase (NDK), an enzyme involved in nucleotide phosphorylation, with progressively restricted amino acid repertoires. They evaluated enzyme activity and reaction products using biochemical assays, high-performance liquid chromatography (HPLC), and mass spectrometry. X-ray crystallography, molecular dynamics simulations, and mutational analyses were used to investigate protein structure, nucleotide binding, and potential catalytic mechanisms.

The findings were interesting. One variant, Arc1-12KR, retained the overall NDK protein fold despite lacking the catalytic histidine typically required for its conventional activity. Unexpectedly, this simplified enzyme converted two molecules of adenosine diphosphate (ADP) into one molecule each of adenosine triphosphate (ATP) and adenosine monophosphate (AMP). HPLC analysis confirmed the formation of both products. Further analyses indicated that aspartate and arginine residues, together with magnesium ions (Mg²⁺), may have helped position the nucleotide molecules and facilitate this alternative phosphoryl-transfer reaction.

Molecular dynamics simulations also indicated that the proposed binding site could accommodate two ADP molecules. However, the precise catalytic mechanism remains unresolved, and alternative pathways cannot be excluded. Arc1-12KR showed an ATP-forming turnover rate of approximately 1.5 s⁻¹. This is about 7,500 times higher than the apparent turnover number reported for short p-loop prototype polypeptides that catalyze the same ADP disproportionation reaction. 

Reflecting on the broader significance, Akanuma noted, “Our finding that a simplified ancestral enzyme can form ATP from ADP suggests that early proteins with limited amino acid compositions may have supported primitive energy metabolism.” Although the findings do not establish that this particular enzyme existed on early Earth, they provide an experimental model for exploring how simpler proteins may have supported energy-related chemistry. The findings could also inform the design of artificial enzymes with reduced amino acid repertoires, potentially helping researchers identify new catalytic activities and develop minimal protein catalysts for biotechnology applications.

Overall, the study shows that restricting amino acid diversity can reveal catalytic capabilities beyond a protein’s conventional function. By demonstrating that a simplified ancestral enzyme can form ATP from ADP, the research offers a new way to investigate how early proteins may have contributed to the evolution of biological energy metabolism.

Reference

Authors: Sota Yagi^1,2, Subrata Dasgupta², Shunsuke Tagami², and Satoshi Akanuma¹
Title of original paper: Switching ATP-Forming Phosphoryl-Transfer Chemistry With a Restricted Set of Amino Acids
Journal: Angewandte Chemie International Edition
DOI: doi.org/10.1002/anie.2166480
Affiliations: 1 Faculty of Human Sciences, Waseda University, Japan
　　　　　 2 RIKEN Center for Integrative Medical Sciences, Japan

Related Link

・Press release of Waseda University

The post Ancestral Enzyme Redesigned with Fewer Amino Acid Types Reveals New Catalytic Possibilities first appeared on Astrobiology Center, NINS.

The findings were posted in Nature Communications on July 27, 2026.

When plants perceive drought stress, they synthesize a key phytohormone called abscisic acid (ABA). ABA basically tells the plants to close their stoma (similar to pores) to reduce water loss. While this defense mechanism is incredibly helpful at conserving water, ABA also mediates some unwanted responses, such as seed dormancy and growth inhibition.

“We want the plants to conserve water to improve survivability, but we don’t want them to suddenly stop growing,” explains Nobuyuki Uozumi (Tohoku University). “To achieve this ideal outcome, we devised a strategy of identifying compounds that inhibit molecules promoting stomatal opening.”The research team focused on the stomata in a model plant (Arabidopsis thaliana). Stomata are partially controlled by a channel called KAT1, which triggers stomatal opening by adjusting how much K⁺ uptake occurs. In other words, KAT1 is trying to keep the doors open – not a good strategy during a drought. The researchers aimed to find KAT1 inhibitors to keep the door shut tight.

To identify inhibitors of KAT1, they performed an electrophysiological chemical screen and identified the small molecule NS5806. They then synthesized a derivative, UA49, by modifying its chemical structure. Application of either compound to leaf epidermal strips successfully induced stomatal closure and inhibited stomatal opening. Moreover, foliar application of NS5806 or UA49 enhanced drought tolerance in plants. Importantly, unlike ABA, neither compound caused undesirable side effects, such as delayed seed germination or inhibited root growth, highlighting their potential for agricultural applications like biostimulant.In addition, the team elucidated the molecular mechanisms underlying the stomatal response induced by NS5806/UA49 – which was distinct from ABA. They compared normal plants to plants that were modified to lack KAT1 channels and looked at intracellular Ca²⁺ concentrations—a key mediator in signaling. In normal plants, there was a sustained influx of Ca²⁺ in the guard cells that open and close stomata. In plants without KAT1 (our notorious door-opener), this response was absent. These findings suggest that the regulation of K⁺ channel activity is intrinsically involved in the modulation of intracellular Ca²⁺ signaling, pointing to a novel, previously unreported possibility that K⁺ channels act as signaling mediators.

The international research team found two compounds that confer drought tolerance with fewer side effects than ABA, making them promising candidates for improving drought tolerance in major crops. The underlying mechanism was also investigated, further highlighting the details of how this strategy could potentially be applied. As climate change makes extreme drought a more common occurrence worldwide, further research in this area is crucial to help ensure that our food supply doesn’t just wither away.

Publication Details

Title: Synthetic ion channel inhibitors enhance plant drought tolerance
Authors: Kanane Sato, Kyota Suzuki, Shunya Saito, Taishin Kakei, Megumi Kato, Masana Yazaki, Yasutaka Kawai, Mieko Arisawa, Nobuhisa Isaka, Toshio Yamaguchi, Matteo Grenzi, Laura Luoni, Masaru Kono, Yuki Hayashi, Toshinori Kinoshita, Farhan Aziz, Khurram Bashir, Motoaki Seki, Asuka Kamimura, Takumi Higaki, Jun Takeuchi, Yasushi Todoroki, Huifei Yin, Francisco Rubio, Jörg Kudla, Shintaro Munemasa, Yoshiyuki Murata, Masaru Tsujii, Yasuhiro Ishimaru, Alex Costa & Nobuyuki Uozumi*
Journal: Nature Communications
DOI:  10.1038/s41467-026-75894-w

Link

Press release by Tohoku University

The post New Molecule Class Creates Hardy, Drought Tolerant Plants first appeared on Astrobiology Center, NINS.