Resurrection and characterization of ancestral CYP11A1 enzymes

Mitochondrial cytochromes P450 presumably originated from a common microsomal ancestor. However, it is still unknown how ancient mitochondrial P450s were able to retain their oxygenase function following relocation the matrix and later emerged as enzymes specialized for steroid hormone biosynthesis in vertebrates. Here, we used approach of ancestral sequence reconstruction (ASR) resurrect CYP11A1 characterize unique biochemical properties. Two variants, CYP11A_Mammal_N101 CYP11A_N1, well an extant bovine form recombinantly expressed purified homogeneity. All showed characteristic spectral properties convert cholesterol other sterol substrates pregnenolone, yet with different specificities. The vertebrate CYP11A_N1 ancestor preferred precursor, desmosterol, substrate suggesting convergent evolution early metabolism enzymes. Both ancestors withstand increased levels hydrogen peroxide but only thermostability (˜ 25 °C increase T 50 ) compared CYP11A1. extraordinary robustness P450s, demonstrated may have allowed them stay active when presented poorly compatible electron transfer proteins resulting harmful ROS new environment matrix. To best our knowledge, this work represents first study that describes resurrection results will help understand gain fundamental functional insights into evolutionary origins animals.