Ancestral Sequence Reconstruction of a Cytochrome P450 Family Involved in Chemical Defense Reveals the Functional Evolution of a Promiscuous, Xenobiotic-Metabolizing Enzyme in Vertebrates

Abstract

The cytochrome P450 family 1 enzymes (CYP1s) are a diverse family of hemoprotein monooxygenases which metabolize many xenobiotics including numerous environmental carcinogens. However, their historical function and evolution remain largely unstudied. Here we investigate CYP1 evolution via the reconstruction and characterization of the vertebrate CYP1 ancestors. Younger ancestors and extant forms generally demonstrated higher activity towards typical CYP1 xenobiotic and steroid substrates than older ancestors, suggesting significant diversification away from the original CYP1 function. Caffeine metabolism appears to be a recently evolved trait of the CYP1A subfamily, observed in the mammalian CYP1A lineage, and may parallel the recent evolution of caffeine synthesis in multiple separate plant species. Likewise, the aryl hydrocarbon receptor agonist, 6-formylindolo[3,2-b]carbazole (FICZ) was metabolised to a greater extent by certain younger ancestors and extant forms, suggesting that activity towards FICZ increased in specific CYP1 evolutionary branches, a process that may have occurred in parallel to the exploitation of land where UV-exposure was higher than in aquatic environments. As observed with previous reconstructions of P450 enzymes, thermostability correlated with evolutionary age; the oldest ancestor was up to 35 °C more thermostable than the extant forms, with a 10T50 (temperature at which 50% of the haemoprotein remains intact after 10 min) of 71 °C. This robustness may have facilitated evolutionary diversification of the CYP1s by buffering the destabilizing effects of mutations that conferred novel functions, a phenomenon which may also be useful in exploiting the catalytic versatility of these ancestral enzymes for commercial application as biocatalysts.

Engineering functional thermostable proteins using ancestral sequence reconstruction

“…This should make it more straightforward to determine the causative changes above the background noise of neutral drift. In our experience, a correlation is generally seen between evolutionary age and thermostability ( 110 ), so choosing ancestors that are more or less closely related should enable specific changes to be identified, by restricting the number of differences between the forms in question. However, to date, insufficient studies have been performed for the full benefit of this approach to be realized.…”

Opportunities for Accelerating Drug Discovery and Development by Using Engineered Drug-Metabolizing Enzymes

“…Studies in the author's laboratory showed DNA shuffling could be used to develop libraries of biocatalysts with varied regioselectivity that could be mined for forms with desired properties, e.g., enhanced production of minor metabolites or novel activities (Huang et al, 2007;Johnston et al, 2007;Hunter et al, 2011;Behrendorff et al, 2013). More recently, ancestral sequence reconstruction has been applied to enhance the thermostability and solvent tolerance of drug-metabolizing P450s and to access forms with altered catalytic properties (Gumulya et al, 2018;Gumulya et al, 2019;Harris et al, 2022;Thomson et al, unpublished).…”

Heme Pocket Structure and Its Functional Implications in an Ancestral Globin Protein

“…For example, ASR was used to develop more stable and robust protein scaffolds for a P450 family. 5 The ancestral enzymes, with improved thermal stability, were expressed at relatively high levels in Escherichia coli (E. coli). Another example is the analysis of an allosteric network of tryptophan synthase composed of two α and two β subunits.…”

“…This information can be used to determine which mutated residues are important in the expression of protein function or to design thermostable proteins. − Although it is difficult to improve the stability of an enzyme using rational methods, ASR has been used to generate active and thermally stable proteins. For example, ASR was used to develop more stable and robust protein scaffolds for a P450 family . The ancestral enzymes, with improved thermal stability, were expressed at relatively high levels in Escherichia coli (E.…”