Ancestral enzyme redesigned with fewer amino acid types reveals new catalytic possibilities

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.

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Reference

Authors: Sota Yagi ^1,2, Subrata Dasgupta², Shunsuke Tagami², and Satoshi Akanuma¹

DOI: doi.org/10.1002/anie.2166480

Affiliations:        

¹Faculty of Human Sciences, Waseda University, Japan

²RIKEN Center for Integrative Medical Sciences, Japan

About Waseda University

Located in the heart of Tokyo, Waseda University is a leading private research university that has long been dedicated to academic excellence, innovative research, and civic engagement at both the local and global levels since 1882. The University has produced many changemakers in its history, including eight prime ministers and many leaders in business, science and technology, literature, sports, and film. Waseda has strong collaborations with overseas research institutions and is committed to advancing cutting-edge research and developing leaders who can contribute to the resolution of complex, global social issues. The University has set a target of achieving a zero-carbon campus by 2032, in line with the Sustainable Development Goals (SDGs) adopted by the United Nations in 2015. 

To learn more about Waseda University, visit https://www.waseda.jp/top/en  

About Professor Satoshi Akanuma

Professor Satoshi Akanuma, PhD, is affiliated with the Faculty of Human Sciences at Waseda University, Japan. He earned his Doctor of Science degree from the Tokyo Institute of Technology in 1998 and has over 25 years of research experience. His research focuses on ancestral protein reconstruction, molecular evolution, enzyme engineering, and the origin of life. His research has contributed to our understanding of protein evolution and the origins of life through research on ancestral enzyme reconstruction and the functional capabilities of early proteins with limited amino acid repertoires.