Resurrection and characterization of ancestral CYP11A1 enzymes

Abstract

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

Single mutations toggle the substrate selectivity of multifunctional Camptotheca secologanic acid synthases

“…Although the use of ASR to identify key developments in CYP specialization is well reported for animal CYPs (39)(40)(41)(42), our work highlights the usefulness of ASR and related methodologies for investigating CYP specialization in plant biosynthetic pathways. In light of the accumulating knowledge of plant specialized metabolism, the large number of CYPs in these pathways, and the burgeoning collections of genomic resources, ASR is emerging as a powerful tool to investigate how particular enzymes and the biosynthetic pathways they constitute have evolved through time.…”

Engineering functional thermostable proteins using ancestral sequence reconstruction

“…Studies using the ancestral mutation method have assessed the stabilizing effect of one or a few ancestral residues and revealed hydrophobic packing, hydrogen bonding and the formation of ion pairs (39,44,47,56,57,99) to be important. In other mutagenesis studies residues in the ancestral proteins have been altered to abolish stability as a way of identifying important interactions (47,66,114). Thus, analysis of ancestral structures has provided complementary information to studies on proteins from thermophiles and underscored the importance of a variety of stabilizing interactions.…”

“…Different colors represent individual studies and for each phylogeny, directly related lineages are connected by solid lines. Sources in the order shown in the figure are: ( 41 , 42 , 43 , 45 , 47 , 48 , 49 , 63 , 64 , 97 , 109 , 115 , 116 , 117 , 118 , 119 , 120 , 121 , 122 ). …”

“…Studies using the ancestral mutation method have assessed the stabilizing effect of one or a few ancestral residues and revealed hydrophobic packing, hydrogen bonding, and the formation of ion pairs ( 39 , 44 , 47 , 51 , 52 , 54 ) to be important. In other mutagenesis studies, residues in the ancestral proteins have been altered to abolish stability as a way of identifying important interactions ( 47 , 63 , 109 ).…”

Ancestral Sequence Reconstruction Enhances Gene Mining Efforts for Industrial Ene Reductases by Expanding Enzyme Panels with Thermostable Catalysts

“…Previous efforts to evaluate ancestral enzyme thermostability showed that ASR-sourced enzymes were associated with higher melting temperatures compared to their WT counterparts. However, in the majority of studies, this conclusion was drawn when comparing only a handful of WT and ASR enzymes ranging from 1 to 8 ASR catalysts. − ,− ,,, Therefore, while it has been well documented in the literature that ASR can produce stabilized enzymes, it has not been clear how statistically significant this effect is. To our knowledge, the work presented here is the largest study that compares thermostability between a balanced number WT and ASR enzymes, 57 and 56, respectively.…”