Resurrection and characterization of ancestral CYP11A1 enzymes

Resurrection and characterization of ancestral CYP11A1 enzymes

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...

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Bibliographic Details
Published in:The FEBS journal Vol. 288; no. 22; pp. 6510 - 6527
Main Authors: Hartz, Philip, Strohmaier, Silja J., EL‐Gayar, Basma M., Abdulmughni, Ammar, Hutter, Michael C., Hannemann, Frank, Gillam, Elizabeth M. J., Bernhardt, Rita
Format: Journal Article
Language:English
Published: Oxford Blackwell Publishing Ltd 01-11-2021
Subjects:
ancestral sequence reconstruction
Biosynthesis
Cholesterol
Cytochromes P450
Electron transfer
Enzymes
Homogeneity
Hydrogen peroxide
Lipid metabolism
Mitochondria
Oxygenase
Pregnenolone
pregnenolone formation
Relocation
steroid hormone biosynthesis
Steroids
Substrates
Thermal stability
Vertebrates
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Summary: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 T50) 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. The mitochondrial CYP11A subfamily enzymes catalyze the side‐chain cleavage of cholesterol. Ancestral sequence reconstruction was used to study the evolution of these cytochrome P450 enzymes, and two ancestral forms were resurrected: The first was the whole subfamily ancestor and the second was the mammalian ancestor. The whole subfamily ancestor showed a high degree of thermal stability and greater relative tolerance to reactive oxygen species than the extant enzyme.
Bibliography:Philip Hartz and Silja J. Strohmaier contributed equally to this article
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ISSN:1742-464X
1742-4658
DOI:10.1111/febs.16054