Sequence selection by FitSS4ASR alleviates ancestral sequence reconstruction as exemplified for geranylgeranylglyceryl phosphate synthase

Abstract For evolutionary studies, but also for protein engineering, ancestral sequence reconstruction (ASR) has become an indispensable tool. The first step of every ASR protocol is the preparation of a representative sequence set containing at most a few hundred recent homologs whose composition determines decisively the outcome of a reconstruction. A common approach for sequence selection consists of several rounds of manual recompilation that is driven by embedded phylogenetic analyses of the varied sequence sets. For ASR of a geranylgeranylglyceryl phosphate synthase, we additionally utilized FitSS4ASR, which replaces this time-consuming protocol with an efficient and more rational approach. FitSS4ASR applies orthogonal filters to a set of homologs to eliminate outlier sequences and those bearing only a weak phylogenetic signal. To demonstrate the usefulness of FitSS4ASR, we determined experimentally the oligomerization state of eight predecessors, which is a delicate and taxon-specific property. Corresponding ancestors deduced in a manual approach and by means of FitSS4ASR had the same dimeric or hexameric conformation; this concordance testifies to the efficiency of FitSS4ASR for sequence selection. FitSS4ASR-based results of two other ASR experiments were added to the Supporting Information. Program and documentation are available at https://gitlab.bioinf.ur.de/hek61586/FitSS4ASR.

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The molecular signal for the adaptation to cold temperature during early life on Earth

Biology Letters ◽

10.1098/rsbl.2013.0608 ◽

2013 ◽

Vol 9 (5) ◽

pp. 20130608 ◽

Cited By ~ 12

Author(s):

Mathieu Groussin ◽

Bastien Boussau ◽

Sandrine Charles ◽

Samuel Blanquart ◽

Manolo Gouy

Keyword(s):

Phylogenetic Trees ◽

Large Scale ◽

Common Ancestor ◽

Phylogenetic Signal ◽

Optimal Growth ◽

Ancestral Sequence ◽

Rate Variation ◽

Last Common Ancestor ◽

Ancestral Sequence Reconstruction ◽

Sequence Reconstruction

Several lines of evidence such as the basal location of thermophilic lineages in large-scale phylogenetic trees and the ancestral sequence reconstruction of single enzymes or large protein concatenations support the conclusion that the ancestors of the bacterial and archaeal domains were thermophilic organisms which were adapted to hot environments during the early stages of the Earth. A parsimonious reasoning would therefore suggest that the last universal common ancestor (LUCA) was also thermophilic. Various authors have used branch-wise non-homogeneous evolutionary models that better capture the variation of molecular compositions among lineages to accurately reconstruct the ancestral G + C contents of ribosomal RNAs and the ancestral amino acid composition of highly conserved proteins. They confirmed the thermophilic nature of the ancestors of Bacteria and Archaea but concluded that LUCA, their last common ancestor, was a mesophilic organism having a moderate optimal growth temperature. In this letter, we investigate the unknown nature of the phylogenetic signal that informs ancestral sequence reconstruction to support this non-parsimonious scenario. We find that rate variation across sites of molecular sequences provides information at different time scales by recording the oldest adaptation to temperature in slow-evolving regions and subsequent adaptations in fast-evolving ones.

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Faculty Opinions recommendation of An experimental phylogeny to benchmark ancestral sequence reconstruction.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.726740118.793524093 ◽

2016 ◽

Author(s):

Reinhard Sterner ◽

Rainer Merkl

Keyword(s):

Ancestral Sequence ◽

Ancestral Sequence Reconstruction ◽

Sequence Reconstruction

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Ancestral sequence reconstruction for protein engineers

Current Opinion in Structural Biology ◽

10.1016/j.sbi.2021.04.001 ◽

2021 ◽

Vol 69 ◽

pp. 131-141

Author(s):

Matthew A. Spence ◽

Joe A. Kaczmarski ◽

Jake W. Saunders ◽

Colin J. Jackson

Keyword(s):

Ancestral Sequence ◽

Ancestral Sequence Reconstruction ◽

Sequence Reconstruction

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Ancestral sequence reconstruction produces thermally stable enzymes with mesophilic enzyme-like catalytic properties

Scientific Reports ◽

10.1038/s41598-020-72418-4 ◽

2020 ◽

Vol 10 (1) ◽

Cited By ~ 2

Author(s):

Ryutaro Furukawa ◽

Wakako Toma ◽

Koji Yamazaki ◽

Satoshi Akanuma

Keyword(s):

Low Temperature ◽

Catalytic Properties ◽

Amino Acid Sequences ◽

Ancestral Sequence ◽

Kinetic Properties ◽

Reconstruction Technique ◽

Thermally Stable ◽

Ancestral Sequence Reconstruction ◽

Ancestral Sequences ◽

Sequence Reconstruction

Abstract Enzymes have high catalytic efficiency and low environmental impact, and are therefore potentially useful tools for various industrial processes. Crucially, however, natural enzymes do not always have the properties required for specific processes. It may be necessary, therefore, to design, engineer, and evolve enzymes with properties that are not found in natural enzymes. In particular, the creation of enzymes that are thermally stable and catalytically active at low temperature is desirable for processes involving both high and low temperatures. In the current study, we designed two ancestral sequences of 3-isopropylmalate dehydrogenase by an ancestral sequence reconstruction technique based on a phylogenetic analysis of extant homologous amino acid sequences. Genes encoding the designed sequences were artificially synthesized and expressed in Escherichia coli. The reconstructed enzymes were found to be slightly more thermally stable than the extant thermophilic homologue from Thermus thermophilus. Moreover, they had considerably higher low-temperature catalytic activity as compared with the T. thermophilus enzyme. Detailed analyses of their temperature-dependent specific activities and kinetic properties showed that the reconstructed enzymes have catalytic properties similar to those of mesophilic homologues. Collectively, our study demonstrates that ancestral sequence reconstruction can produce a thermally stable enzyme with catalytic properties adapted to low-temperature reactions.

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