scholarly journals Analysis of allosteric communication in a multienzyme complex by ancestral sequence reconstruction

2019 ◽  
Vol 117 (1) ◽  
pp. 346-354 ◽  
Author(s):  
Michael Schupfner ◽  
Kristina Straub ◽  
Florian Busch ◽  
Rainer Merkl ◽  
Reinhard Sterner
Keyword(s):  
Community Analysis ◽  
Signal Propagation ◽  
Ancestral Sequence ◽  
Site Directed Mutagenesis ◽  
Tryptophan Synthase ◽  
Α Subunit ◽  
Ancestral Sequence Reconstruction ◽  
Multienzyme Complexes ◽  
Sequence Reconstruction ◽  
Dynamics Simulations

Tryptophan synthase (TS) is a heterotetrameric αββα complex. It is characterized by the channeling of the reaction intermediate indole and the mutual activation of the α-subunit TrpA and the β-subunit TrpB via a complex allosteric network. We have analyzed this allosteric network by means of ancestral sequence reconstruction (ASR), which is an in silico method to resurrect extinct ancestors of modern proteins. Previously, the sequences of TrpA and TrpB from the last bacterial common ancestor (LBCA) have been computed by means of ASR and characterized. LBCA-TS is similar to modern TS by forming a αββα complex with indole channeling taking place. However, LBCA-TrpA allosterically decreases the activity of LBCA-TrpB, whereas, for example, the modern ncTrpA fromNeptuniibacter caesariensisallosterically increases the activity of ncTrpB. To identify amino acid residues that are responsible for this inversion of the allosteric effect, all 6 evolutionary TrpA and TrpB intermediates that stepwise link LBCA-TS with ncTS were characterized. Remarkably, the switching from TrpB inhibition to TrpB activation by TrpA occurred between 2 successive TS intermediates. Sequence comparison of these 2 intermediates and iterative rounds of site-directed mutagenesis allowed us to identify 4 of 413 residues from TrpB that are crucial for its allosteric activation by TrpA. The effect of our mutational studies was rationalized by a community analysis based on molecular dynamics simulations. Our findings demonstrate that ancestral sequence reconstruction can efficiently identify residues contributing to allosteric signal propagation in multienzyme complexes.

Ancestral sequence reconstruction for protein engineers

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

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

2020 ◽  
Vol 10 (1) ◽  
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.

scholarly journals Alignment Modulates Ancestral Sequence Reconstruction Accuracy

2018 ◽  
Vol 35 (7) ◽  
pp. 1783-1797 ◽  
Author(s):  
Ricardo Assunção Vialle ◽  
Asif U Tamuri ◽  
Nick Goldman
Keyword(s):  
Ancestral Sequence ◽  
Reconstruction Accuracy ◽  
Ancestral Sequence Reconstruction ◽  
Sequence Reconstruction

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

2019 ◽  
Vol 400 (3) ◽  
pp. 367-381 ◽  
Author(s):  
Kristina Straub ◽  
Mona Linde ◽  
Cosimo Kropp ◽  
Samuel Blanquart ◽  
Patrick Babinger ◽  
...  
Keyword(s):  
Phylogenetic Signal ◽  
Phylogenetic Analyses ◽  
Specific Property ◽  
Ancestral Sequence ◽  
Rational Approach ◽  
Ancestral Sequence Reconstruction ◽  
Representative Sequence ◽  
Sequence Selection ◽  
Sequence Reconstruction ◽  
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.

scholarly journals Ancestral Sequence Reconstruction in Primate Mitochondrial DNA: Compositional Bias and Effect on Functional Inference

2004 ◽  
Vol 21 (10) ◽  
pp. 1871-1883 ◽  
Author(s):  
Neeraja M. Krishnan ◽  
Hervé Seligmann ◽  
Caro-Beth Stewart ◽  
A. P. Jason de Koning ◽  
David D. Pollock
Keyword(s):  
Mitochondrial Dna ◽  
Ancestral Sequence ◽  
Compositional Bias ◽  
Ancestral Sequence Reconstruction ◽  
Sequence Reconstruction ◽  
Functional Inference
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