Some like it hot, others not
Enzymes strike a delicate balance between features that enhance chemical reactivity and those that contribute to stable structure. Both features are important and can be unrelated or antagonistic. Pinney et al. combined rich experimental work on thermophilic and mesophilic variants of the enzyme ketosteroid isomerase (KSI) with bioinformatic data from a diverse set of bacterial enzymes to reveal the molecular determinants of thermal adaptation in enzymes. For KSI, they observed a trade-off between activity and thermal stability that comes down to a single active-site residue. With their larger dataset, they identified patterns of individual amino acid substitutions that are favored at higher temperatures, and also consider how networks of stabilizing interactions develop.
Science, this issue p. eaay2784
Over billions of years, organisms and their enzymes have been evolving and adapting in response to selection pressures from their environments. In particular, livable temperature varies from about −15° to 121°C and exerts an evolutionary force that manifests in the adaptation of enzyme stabilities and activities: At increased temperatures, enzymes evolve greater stability to combat thermal denaturation and maintain a folded structure, whereas at decreased temperatures, nearly all chemical reactions necessary for life slow, placing evolutionary pressure on cold-adapted enzymes to be more active. An understanding of the molecular and evolutionary mechanisms that underlie enzyme temperature adaptation are integral to our understanding of how living systems have evolved and can reveal hidden aspects of how enzyme activity and stability is achieved, helping to define rules that can be used for enzyme design.
We have dissected the molecular and evolutionary mechanisms underlying enzyme temperature adaptation both deeply and broadly. We first turned to the bacterial enzyme ketosteroid isomerase (KSI), combining mechanistic and structural studies with sequence and phylogenetic analyses to reveal the mechanisms underlying its activity and stability adaptation at the atomic and residue levels. Building on these results, we performed sequence and phylogenetic analyses, examining enzyme temperature adaptation in 2194 bacterial enzyme families to identify residue changes associated with growth temperature differences (referred to as “temperature-associated residues”) and analyzing their physical properties and interactions.
We show that temperature adaptation in KSI arises primarily from a single active-site residue change with minimal epistasis. In cold-adapted KSI orthologs, a stronger active-site hydrogen-bond donor, protonated Asp103 (D103), improves activity. Conversely, warm-adapted KSI orthologs are stabilized by Ser103 (S103), which decreases activity but increases stability by removing the protonation-coupled folding of D103. Phylogenetic analyses showed that this active-site amino acid change (D103/S103) has occurred in diverse KSI sequence backgrounds from diverse bacteria, further supporting limited epistasis and suggesting parallel adaptation.
Our broad sequence and phylogenetic analyses revealed 158,184 statistically significant temperature-associated residues from 1005 enzyme families. Most of these residues are found in sequences from phylogenetically diverse bacteria, suggesting widespread temperature adaptation and parallel evolution. By mapping temperature-associated residues to structure, we found that these residues typically change with temperature on their own or with one other residue in physical contact, suggesting limited epistasis at these sites. Analyses of these temperature-associated residues reveal molecular and physical trends that test, hone, and revise nearly all prior mechanisms for enzyme temperature adaptation and identify networks of residues that appear to coadapt to temperature, perhaps cooperatively influencing catalysis stability, and/or allostery.
Our results broadly and deeply addressed enzyme temperature adaptation, revealing molecular mechanisms underlying the adaptation of KSI and identifying 158,184 temperature-associated residues; these data reveal physical trends and provide extensive data that can be further mined to understand molecular evolution and applied to enzyme design. These data further suggest that enzyme adaptation has repeatedly followed evolutionary paths of low epistasis, advancing our understanding of the evolutionary mechanisms that underly adaptation of nature’s repertoire of enzymes.
The mechanisms that underly the adaptation of enzyme activities and stabilities to temperature are fundamental to our understanding of molecular evolution and how enzymes work. Here, we investigate the molecular and evolutionary mechanisms of enzyme temperature adaption, combining deep mechanistic studies with comprehensive sequence analyses of thousands of enzymes. We show that temperature adaptation in ketosteroid isomerase (KSI) arises primarily from one residue change with limited, local epistasis, and we establish the underlying physical mechanisms. This residue change occurs in diverse KSI backgrounds, suggesting parallel adaptation to temperature. We identify residues associated with organismal growth temperature across 1005 diverse bacterial enzyme families, suggesting widespread parallel adaptation to temperature. We assess the residue properties, molecular interactions, and interaction networks that appear to underly temperature adaptation.