scholarly journals Structural Analysis of the Ancestral Haloalkane Dehalogenase AncLinB-DmbA

2021 ◽  
Vol 22 (21) ◽  
pp. 11992
Author(s):  
Andrii Mazur ◽  
Pavel Grinkevich ◽  
Radka Chaloupkova ◽  
Petra Havlickova ◽  
Barbora Kascakova ◽  
...  
Keyword(s):  
Cell Biology ◽  
Environmental Pollutants ◽  
Hydrolytic Degradation ◽  
Ancestral Sequence ◽  
Diffusion Method ◽  
X Rays ◽  
Molecular Tagging ◽  
Ancestral Sequence Reconstruction ◽  
Active Site Cavity ◽  
Sequence Reconstruction

Haloalkane dehalogenases (EC 3.8.1.5) play an important role in hydrolytic degradation of halogenated compounds, resulting in a halide ion, a proton, and an alcohol. They are used in biocatalysis, bioremediation, and biosensing of environmental pollutants and also for molecular tagging in cell biology. The method of ancestral sequence reconstruction leads to prediction of sequences of ancestral enzymes allowing their experimental characterization. Based on the sequences of modern haloalkane dehalogenases from the subfamily II, the most common ancestor of thoroughly characterized enzymes LinB from Sphingobium japonicum UT26 and DmbA from Mycobacterium bovis 5033/66 was in silico predicted, recombinantly produced and structurally characterized. The ancestral enzyme AncLinB-DmbA was crystallized using the sitting-drop vapor-diffusion method, yielding rod-like crystals that diffracted X-rays to 1.5 Å resolution. Structural comparison of AncLinB-DmbA with their closely related descendants LinB and DmbA revealed some differences in overall structure and tunnel architecture. Newly prepared AncLinB-DmbA has the highest active site cavity volume and the biggest entrance radius on the main tunnel in comparison to descendant enzymes. Ancestral sequence reconstruction is a powerful technique to study molecular evolution and design robust proteins for enzyme technologies.

The tetrameric structure of the novel haloalkane dehalogenase DpaA from Paraglaciecola agarilytica NO2

2021 ◽  
Vol 77 (3) ◽  
pp. 347-356
Author(s):  
Andrii Mazur ◽  
Tatyana Prudnikova ◽  
Pavel Grinkevich ◽  
Jeroen R. Mesters ◽  
Daria Mrazova ◽  
...  
Keyword(s):  
Cell Biology ◽  
Environmental Pollutants ◽  
Gel Filtration ◽  
Halophilic Bacterium ◽  
Size Exclusion ◽  
Diffusion Method ◽  
X Rays ◽  
The Novel ◽  
Haloalkane Dehalogenase ◽  
Tetrameric Structure

Haloalkane dehalogenases (EC 3.8.1.5) are microbial enzymes that catalyse the hydrolytic conversion of halogenated compounds, resulting in a halide ion, a proton and an alcohol. These enzymes are used in industrial biocatalysis, bioremediation and biosensing of environmental pollutants or for molecular tagging in cell biology. The novel haloalkane dehalogenase DpaA described here was isolated from the psychrophilic and halophilic bacterium Paraglaciecola agarilytica NO2, which was found in marine sediment collected from the East Sea near Korea. Gel-filtration experiments and size-exclusion chromatography provided information about the dimeric composition of the enzyme in solution. The DpaA enzyme was crystallized using the sitting-drop vapour-diffusion method, yielding rod-like crystals that diffracted X-rays to 2.0 Å resolution. Diffraction data analysis revealed a case of merohedral twinning, and subsequent structure modelling and refinement resulted in a tetrameric model of DpaA, highlighting an uncommon multimeric nature for a protein belonging to haloalkane dehalogenase subfamily I.

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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