Biology subjects

Akanuma, S.

Publications and source records attributed to Akanuma, S..

3 recordsLinked to original sources

bioRxiv2026

Emergence of a potentially ancestral ATP-synthesizing activity under prebiotic amino acid constraints

How amino acid composition shapes enzymatic function remains a central question in molecular evolution. Early proteins were likely composed of a limited set of amino acids, yet the catalytic properties of proteins under such compositional constraints are still poorly understood. Here we show that restricting amino acid repertoires can replace native enzymatic activity with an alternative phosphoryl-transfer reaction that disproportionates ADP into ATP and AMP. Using a reconstructed ancestral nucleoside diphosphate kinase (NDK) variant composed of a restricted amino acid set, we demonstrate this reaction, which is not observed in extant NDKs but is chemically analogous to that catalyzed by adenylate kinase. Although modest in catalytic rate, X-ray crystallography, molecular dynamics, and mutational analyses reveal a distinct active-site organization in which aspartate and arginine residues cooperatively coordinate Mg2+ and support phosphoryl transfer through a noncanonical mechanism. Lysine can substitute for arginine under these constraints while retaining activity. Together, these findings show that restricting amino acid diversity can remodel active sites and promote alternative phosphoryl-transfer reactions, illustrating how limitations in amino acid availability could influence catalytic functions in early enzyme evolution. Significant StatementThe first enzymes arose on early Earth, where only a limited set of amino acids was likely available. Whether such limitations reduce catalytic efficiency or reshape enzyme function is still unclear. We reconstructed a model of an ancestral enzyme using a simplified amino acid set approximating those available on early Earth and found that restricting amino acid diversity replaced its original catalytic activity with an alternative function that produces ATP from two ADP molecules. Structural and mutational analyses indicate that this shift involves a reorganization of the active site. These results provide experimental evidence that limiting amino acid diversity can reshape catalytic function and may have influenced the emergence of catalytic activities during enzyme evolution.

evolutionary biology↗

bioRxiv2025

Functional and Structural Characterization of F1-ATPase with common ancestral core domains in stator ring

Extant F1-ATPases exhibit diverse rotational stepping behaviors--3-, 6-, or 9-step cycles--yet the evolutionary origin of these patterns remains unclear. Here, we used ancestral sequence reconstruction to infer the catalytic {beta} and non-catalytic subunits of a putative ancestral F1-ATPase. We then fused their functionally critical domains into the thermostable F1 from Bacillus PS3, yielding a stable chimeric enzyme. Cryo-EM revealed two distinct conformational states--binding and catalytic dwell states--separated by a [~]34{degrees} rotation of the {gamma} subunit, suggesting a fundamental six-step mechanism akin to that of extant 6-stepping F1-ATPases. Single-molecule rotation assays with ATP and the slowly hydrolyzed ATP analog ATP{gamma}S demonstrated that the chimeric motor is intrinsically a 6-stepper, pausing at binding and catalytic dwell positions separated by 32.1{degrees}, although the binding dwell is significantly prolonged by an unknown mechanism. These findings indicate that F1-ATPase was originally a 6-stepper and diversified into 3-, 6- and 9-step forms in evolutional adaptation. Based on these results, we discuss plausible features of the entire FoF1 complex, along with potential physiological contexts in last universal common ancestor and related lineages.

biophysics↗

bioRxiv2024

Insights into the low-temperature adaptation of an enzyme as studied through ancestral sequence reconstruction

For billions of years, enzymes have evolved in response to the changing environments in which their host organisms lived. Various lines of evidence suggest the earliest primitive organisms inhabited high-temperature environments and possessed enzymes adapted to such conditions. Consequently, extant mesophilic and psychrophilic enzymes are believed to have adapted to lower temperatures during the evolutionary process. Herein, we analyzed this low-temperature adaptation using ancestral sequence reconstruction. Previously, we generated the phylogenetic tree of 3-isopropylmalate dehydrogenases (IPMDHs) and reconstructed the sequence of the last bacterial common ancestor. The corresponding ancestral enzyme displayed high thermostability and catalytic activity at elevated temperatures but moderate activity at low temperatures (Furukawa et al., Sci. Rep. 10, 15493 (2020)). Here, to identify amino acid residues that are responsible for the low-temperature adaptation, we reconstructed and characterized all eleven evolutionary intermediates that sequentially connect the last bacterial common ancestor with extant mesophilic IPMDH from Escherichia coli. A remarkable change in catalytic properties, from those suited for high reaction temperatures to those adapted for low temperatures, occurred between two consecutive evolutionary intermediates. Using a combination of sequence comparisons between ancestral proteins and site-directed mutagenesis analyses, three key amino acid substitutions were identified that enhance low-temperature catalytic activity. Intriguingly, amino acid substitutions that had the most significant impact on activity at low temperatures displayed no discernable effect on thermostability. However, these substitutions markedly reduced the activation energy for catalysis, thereby improving low-temperature activity. Our findings exemplify how ancestral sequence reconstruction can identify residues crucial for adaptation to low temperatures.

evolutionary biology↗