The characterization of ancient Methanococcales malate dehydrogenases reveals that strong thermal stability prevents unfolding under intense γ-irradiation

Malate dehydrogenases (MalDHs) (EC.1.1.1.37), which are involved in the conversion of oxaloacetate to pyruvate in the tricarboxylic acid cycle, are a relevant model for the study of enzyme evolution and adaptation. Likewise, a recent study showed that Methanococcales, a major lineage of Archaea, is a good model to study the molecular processes of proteome thermoadaptation in prokaryotes. Here, we use ancestral sequence reconstruction and paleoenzymology to characterize both ancient and extant MalDHs. We observe a good correlation between inferred optimal growth temperatures and experimental optimal temperatures for activity (A-Topt). In particular, we show that the MalDH present in the ancestor of Methanococcales was hyperthermostable and had an A-Topt of 80 °C, consistent with a hyperthermophilic lifestyle. This ancestor gave rise to two lineages with different thermal constraints: one remained hyperthermophilic, while the other underwent several independent adaptations to colder environments. Surprisingly, the enzymes of the first lineage have retained a thermoresistant behavior (i.e. strong thermostability and high A-Topt), whereas the ancestor of the second lineage shows a strong thermostability, but a reduced A-Topt. Using mutants, we mimic the adaptation trajectory toward mesophily and show that it is possible to significantly reduce the A-Topt without altering the thermostability of the enzyme by introducing a few mutations. Finally, we reveal an unexpected link between thermostability and the ability to resist γ-irradiation-induced unfolding.