How to resurrect ancestral proteins as proxies for ancient biogeochemistry.

Throughout the history of life, enzymes have served as the primary molecular mediators of biogeochemical cycles by catalyzing the metabolic pathways that interact with geochemical substrates. The byproducts of enzymatic activities have been preserved as chemical and isotopic signatures in the geologic record. However, interpretations of these signatures are limited by the assumption that such enzymes have remained functionally conserved over billions of years of molecular evolution. By reconstructing ancient genetic sequences in conjunction with laboratory enzyme resurrection, preserved biogeochemical signatures can instead be related to experimentally constrained, ancestral enzymatic properties. We may thereby investigate instances within molecular evolutionary trajectories potentially tied to significant biogeochemical transitions evidenced in the geologic record. Here, we survey recent enzyme resurrection studies to provide a reasoned assessment of areas of success and common pitfalls relevant to ancient biogeochemical applications. We conclude by considering the Great Oxidation Event, which provides a constructive example of a significant biogeochemical transition that warrants investigation with ancestral enzyme resurrection. This event also serves to highlight the pitfalls of facile interpretation of paleophenotype models and data, as applied to two examples of enzymes that likely both influenced and were influenced by the rise of atmospheric oxygen - RuBisCO and nitrogenase.