Engineering indel and substitution variants of diverse and ancient enzymes using Graphical Representation of Ancestral Sequence Predictions (GRASP)

Foley, Gabriel; Mora, Ariane; Ross, Connie M.; Bottoms, Scott; Sützl, Leander; Lamprecht, Marnie L.; Zaugg, Julian; Essebier, Alexandra; Balderson, Brad; Newell, R. B.; Thomson, Raine E. S.; Kobe, Boštjan; Barnard, Ross; Guddat, Luke W.; Schenk, Gerhard; Carsten, Jörg; Gumulya, Yosephine; Rost, Burkhard; Haltrich, Dietmar; Sieber, Volker; Gillam, Elizabeth M. J.; Bodén, Mikael

doi:10.1101/2019.12.30.891457

Cited by 24 publications

(22 citation statements)

References 58 publications

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“…A total of 339 sequences were included in the final alignment, which was then used to generate the phylogenetic tree with the RaxML program [60]. The GRASP web server was used for the ancestral inference [61]. The N termini of the inferred ancestors were modified according to Wada et al [62] and the N-terminal truncation of inferred sequences was done based on the MSA and the alignment with the bovine CYP11A1 extant form.…”

Section: Ancestral Sequence Reconstructionmentioning

confidence: 99%

Resurrection and characterization of ancestral CYP11A1 enzymes

et al. 2021

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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.

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Section: Ancestral Sequence Reconstructionmentioning

confidence: 99%

Resurrection and characterization of ancestral CYP11A1 enzymes

et al. 2021

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“…Ancestral states for inner nodes for 156, 157, 158, 484 and 494 positions of S protein were inferred using the command line version of GRASP-suite [ 17 ]. This tool for inference was chosen due to its high speed and, most importantly, ability to handle insertions and deletions.…”

Section: Methodsmentioning

confidence: 99%

Determining the International Spread of B.1.1.523 SARS-CoV-2 Lineage with a Set of Mutations Highly Associated with Reduced Immune Neutralization

et al. 2022

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Here, we report the emergence of the variant lineage B.1.1.523 that contains a set of mutations including 156_158del, E484K and S494P in the spike protein. E484K and S494P are known to significantly reduce SARS-CoV-2 neutralization by convalescent and vaccinated sera and are considered as mutations of concern. Lineage B.1.1.523 presumably originated in the Russian Federation and spread across European countries with the peak of transmission in April–May 2021. The B.1.1.523 lineage has now been reported from 31 countries. In this article, we analyze the possible origin of this mutation subset and its immune response using in silico methods.

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“…com/ ramba ut/ figtr ee/ relea ses). Ancestral sequence reconstruction was performed with the FastML software (Ashkenazy et al 2012) and further validated independently using the Graphical Representation of Ancestral Sequence Predictions (GRASP) software (Foley et al 2020). Statistical confidence in each position's reconstructed state for each ancestor was determined from posterior probability; any reconstructed positions with less than 95% posterior probability was considered ambiguous, and alternate states were also tested.…”

Section: Construction Of Spike Glycoprotein Ancestral Sequencementioning

confidence: 99%