Non-Active Site Mutations Drive Enzyme Adaptation to Low Temperatures

Enzymes that originally evolved in high-temperature environments later adapted to cooler conditions as Earth underwent global cooling. Now, researchers from Waseda University and RIKEN in Japan have shown that key evolutionary changes in enzyme function were driven by amino acid substitutions located far from the active site—highlighting a surprising mechanism behind temperature adaptation.

These distant mutations reduced the activation energy required for reactions, improving catalytic efficiency at lower temperatures. The study underscores how large-scale climate events, like global cooling, may have influenced enzyme evolution over time.

Life on Earth has been evolving for billions of years, continuously adapting to environmental shifts. Enzymes—proteins that speed up biochemical reactions inside cells—have evolved alongside their host organisms. Each enzyme has an optimal temperature range for activity. For example, human enzymes perform best around 37 °C (normal body temperature), and their activity drops significantly outside that range.

But not all organisms live in such stable conditions. Extremophiles—organisms that thrive in extreme environments like hot springs or polar regions—possess enzymes finely tuned to these harsh habitats. Thermophilic bacteria, for instance, produce enzymes that remain stable and effective at high temperatures, but those same enzymes tend to lose activity in cooler environments. On the flip side, enzymes from mesophiles and psychrophiles are efficient at moderate to cold temperatures but lack heat tolerance.

Evidence suggests that early life forms were thermophiles, adapting to cooler conditions over time as Earth's climate changed. A key to functioning well at lower temperatures is increased molecular flexibility, but the precise mechanisms behind this adaptation are still being uncovered.

To explore how enzymes from heat-loving organisms evolved to work in cooler environments, researchers used ancestral sequence reconstruction (ASR). ASR combines molecular phylogenetics with genetic and protein engineering to infer and recreate the protein sequences of extinct organisms using data from modern species.

For this study, the team focused on 3-isopropylmalate dehydrogenase (IPMDH), an enzyme involved in leucine biosynthesis. IPMDH is an ideal model for studying enzyme adaptation due to its long evolutionary history and known role in thermostability.

Led by Professor Satoshi Akanuma at Waseda University, in collaboration with Assistant Professor Sota Yagi, Dr. Subrata Dasgupta, and Dr. Shunsuke Tagami from RIKEN, the team traced IPMDH’s evolution from an ancient thermophilic ancestor to the enzyme found in Escherichia coli. Their findings were published in Protein Science.

“We reconstructed 11 intermediate ancestral enzymes along the evolutionary path from the last common bacterial ancestor to E. coli IPMDH,” said Akanuma. “Then we measured how enzyme activity changed at each stage, focusing on improvements in catalytic performance at lower temperatures.”

The team observed a striking jump in activity at 25 °C between two specific ancestors—Anc05 and Anc06. This increase didn’t follow a gradual trend but instead occurred suddenly, suggesting a key evolutionary shift.

To understand what caused this jump, the researchers compared amino acid sequences and used site-directed mutagenesis to introduce targeted mutations. They identified three specific amino acid changes—none of them near the active site—that dramatically boosted enzyme activity at cooler temperatures.

This finding challenges the long-held belief that active site mutations are the main drivers of temperature adaptation. Using molecular dynamics simulations, the researchers discovered that Anc05 retained an open structure, while Anc06 adopted a partially closed conformation. This structural shift lowered the activation energy required for catalysis, making the enzyme more efficient at lower temperatures.

Notably, this transition occurred roughly 2.5 to 2.1 billion years ago—around the time of the Great Oxidation Event, when atmospheric methane levels dropped and global temperatures declined. The team suggests this climatic shift may have pressured enzymes to adapt for better performance in cooler environments.

By pinpointing structural and sequence-level changes that improve enzyme efficiency, ASR offers powerful insights into how life responded to Earth’s evolving climate. According to Akanuma, “Applying this method to other enzymes will help reveal how organisms and their molecular machinery have adapted over the past four billion years.”

Beyond evolutionary biology, these insights could support the engineering of enzymes tailored for industrial, pharmaceutical, and environmental applications—especially those that require reliable performance across different temperature conditions.