Thermostability is the key to maintain the structural integrity and catalytic activity of enzymes in industrial biotechnological processes, such as terpene cyclase-mediated generation of medicines, chiral synthons, and fine chemicals. However, affording a large increase in the thermostability of enzymes through site directed protein engineering techniques can constitute a challenge. In this paper, we used ancestral sequence reconstruction to create a hyperstable variant of the ent-copalyl diphosphate synthase PtmT2, a terpene cyclase involved in the assembly of antibiotics. Molecular dynamics simulations on the its timescale were performed to shed light on possible molecular mechanisms contributing to activity at an elevated temperature and the large 40 degrees C increase in melting temperature observed for an ancestral variant of PtmT2. In silico analysis revealed key differences in the flexibility of a loop capping the active site, between extant and ancestral proteins. For the modern enzyme, the loop collapses into the active site at elevated temperatures, thus preventing biocatalysis, whereas the loop remains in a productive conformation both at ambient and high temperatures in the ancestral variant. Restoring a Pro loop residue introduced in the ancestral variant to the corresponding Gly observed in the extant protein led to reduced catalytic activity at high temperatures, with only moderate effects on the melting temperature, supporting the importance of the flexibility of the capping loop in thermoadaptation. Conversely, the inverse Gly to Pro loop mutation in the modern enzyme resulted in a 3-fold increase in the catalytic rate. Despite an overall decrease in maximal activity of ancestor compared to wild type, its increased thermostability provides a robust backbone amenable for further enzyme engineering. Our work cements the importance of loops in enzyme catalysis and provides a molecular mechanism contributing to thermoadaptation in an ancestral enzyme.

The pandemic caused by Severe acute respiratory syndrome coronavirus 2 has had devastating consequences on global health and economy. Despite the success of vaccination campaigns emerging variants are of concern and novel viruses with the potential to drive future pandemics are circulating in nature. Development of vaccines can be challenging, as key viral protein antigens can be unstable or aggregate. In this study, we present the application of ancestral sequence reconstruction on coronavirus spike protein, resulting in stable and highly soluble ancestral scaffold antigens (AnSAs). The AnSAs interacted with plasma of patients recovered from COVID-19 but did not bind to the human angiotensin-converting enzyme 2 (ACE2) receptor. Cryo-EM analysis of the AnSAs yielded high resolution structures (2.6-2.8 Å) indicating a closed pre-fusion conformation in which all three receptor-binding domains (RBDs) are facing downwards. This captured closed state is stabilised by an intricate hydrogen‑bonding network mediated by well-resolved loops, both within and across monomers, tethering the N‑terminal domain and RBD together, which determines their relative spatial orientation. Finally, we show how AnSAs are potent scaffolds by replacing the ancestral RBD with the Wuhan wild-type sequence, which restored ACE2 binding and increased the interaction with convalescent plasma. In contrast to rational antigen design depending on prior structural knowledge, our work highlights how stable and potentially interesting antigens can be generated using exclusively available sequence information.

The formation of tetracyclic lanosterol from (S)-2,3-oxidosqualene is catalyzed by oxidosqualenecyclase (OSC). Lanosterol is of high interest due to its essential role in steroid metabolism. Therefore,understanding how the inherent high entropic cost of forming a multicyclic core from a flexible linearsubstrate is energetically driven is of high interest. Enzyme mechanisms can involve a reducedhydration state of rearranging transient charges in intermediates and transition states. Often thesereactions have an unusually low entropy barrier. We studied the activation enthalpy and entropy inrelation to solvent tunnels accessing the active site in the carbocationic polycyclization cascadecatalyzed by human OSC (hOSC). We applied Eyring transition state analysis of lanosterol formationby hOSC at different temperatures, alongside Molecular Dynamics simulations and CAVER analysis.hOSC showed a high favorable entropy of activation (+6.4 kcal mol-1 at 310 K) at ambienttemperatures. The introduction of bulky residues at the interface of several water tunnels, resulted inenzyme variants with altered thermodynamic properties. One of the variants was enthalpy-driven andshowed an inversed temperature dependence of cyclization. We further biochemically characterizeddifferent enzyme libraries, in which rational tunnel-mutations combined with mutations suggested byphylogeny-guided protein design were introduced in different combinations. This approach yieldedseveral highly active hOSC variants (5-6x increased activity at 37 °C), as well as a highly active variantat colder temperature with inversed temperature dependence. In summary, the present workhighlights the importance of activation entropy in enzymes, which is often considered negligible, aswell as the challenges associated with rational protein design aiming to modify activationthermodynamic parameters.