This article has been reviewed according to Science X's editorial process and policies. Editors have highlighted the following attributes while ensuring the content's credibility: 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 Oct. 9, 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. 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.
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