Ancestral sequence reconstruction is a powerful method for inferring ancestors of modern enzymes and for studying structure-function relationships of enzymes. We have previously applied this approach to haloalkane dehalogenases (HLDs) from the subfamily HLD-II and obtained thermodynamically highly stabilized enzymes (ΔT m up to 24 °C), showing improved catalytic properties. Here we combined crystallographic structural analysis and computational molecular dynamics simulations to gain insight into the mechanisms by which ancestral HLDs became more robust enzymes with novel catalytic properties. Reconstructed ancestors exhibited similar structure topology as their descendants with the exception of a few loop deviations. Strikingly, molecular dynamics simulations revealed restricted conformational dynamics of ancestral enzymes, which prefer a single state, in contrast to modern enzymes adopting two different conformational states. The restricted dynamics can potentially be linked to their exceptional stabilization. The study provides molecular insights into protein stabilization due to ancestral sequence reconstruction, which is becoming a widely used approach for obtaining robust protein catalysts.

• Klíčová slova
• Ancestral sequence reconstruction, Conformational flexibility, Enzyme, Haloalkane dehalogenase, Protein design, Protein simulations, Thermostability, X-ray crystallography,
• Publikační typ
• časopisecké články MeSH

Enzymes of microbial origin are of immense importance for organic material decomposition leading to bioremediation of organic waste, bioenergy generation, large-scale industrial bioprocesses, etc. The market demand for microbial cellulase enzyme is growing more rapidly which ultimately becomes the driving force towards research on this biocatalyst, widely used in various industrial activities. The use of novel cellulase genes obtained from various thermophiles through metagenomics and genetic engineering as well as following metabolic engineering pathways would be able to enhance the production of thermophilic cellulase at industrial scale. The present review is mainly focused on thermophilic cellulolytic bacteria, discoveries on cellulase gene, genetically modified cellulase, metabolic engineering, and their various industrial applications. A lot of lacunae are yet to overcome for thermophiles such as metagenome analysis, metabolic pathway modification study, search of heterologous hosts in gene expression system, and improved recombinant strain for better cellulase yield as well as value-added product formation.

• MeSH
• Bacteria enzymologie   genetika   metabolismus   MeSH
• celulasa genetika   MeSH
• celulosa metabolismus   MeSH
• enzymy MeSH
• genetické inženýrství MeSH
• metabolické inženýrství * MeSH
• metagenomika MeSH
• průmyslová mikrobiologie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• celulasa MeSH
• celulosa MeSH
• enzymy MeSH

Recombinant mesophilic Escherichia coli (Ec) and thermophilic Bacillus stearothermophilus (Bst) elongation factors EF-Tus, their isolated G-domains, and six chimeric EF-Tus composed of domains of either EF-Tu were prepared, and their GDP/GTP binding activities and thermostability were characterized. BstEF-Tu and BstG-domain bound GDP and GTP with affinities in nanomolar and submicromolar ranges, respectively, fully comparable with those of EcEF-Tu. In contrast, the EcG-domain bound the nucleotides with much lower, micromolar affinities. The exchange of domains 2 and 3 had essentially no effect on the GDP-binding activity; all complexes of chimeric EF-Tus with GDP retained K(d) values in the nanomolar range. The final thermostability level of either EF-Tu was the result of a cooperative interaction between the G-domains and domains 2 + 3. The G-domains set up a "basic" level of the thermostability, which was approximately 20 degrees C higher with the BstG-domain than with the EcG-domain. This correlated with the growth temperature optimum difference of both bacteria and two distinct thermostabilization features of the BstG-domain: an increase of charged residues at the expense of polar uncharged residues (CvP bias), and a decrease in the nonpolar solvent-accessible surface area. Domains 2 + 3 contributed by further stabilization of alpha-helical regions and, in turn, the functions of the G-domains to the level of the respective growth temperature optima. Their contributions were similar irrespective of their origin but, with Ecdomains 2 + 3, dependent on the guanine nucleotide binding state. It was lower in the GTP conformation, and the mechanism involved the destabilization of the alpha-helical regions of the G-domain by Ecdomain 2.

• MeSH
• aminokyseliny chemie   MeSH
• bakteriální proteiny chemie   genetika   metabolismus   MeSH
• cirkulární dichroismus MeSH
• denaturace proteinů MeSH
• druhová specificita MeSH
• elongační faktor Tu chemie   genetika   metabolismus   MeSH
• Escherichia coli chemie   MeSH
• genetická variace MeSH
• Geobacillus stearothermophilus chemie   MeSH
• guanosindifosfát metabolismus   MeSH
• guanosintrifosfát metabolismus   MeSH
• kinetika MeSH
• konformace proteinů MeSH
• molekulární modely MeSH
• proteiny z Escherichia coli chemie   genetika   metabolismus   MeSH
• rekombinantní fúzní proteiny chemie   izolace a purifikace   metabolismus   MeSH
• sbalování proteinů MeSH
• sekundární struktura proteinů MeSH
• teplota MeSH
• terciární struktura proteinů MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• aminokyseliny MeSH
• bakteriální proteiny MeSH
• elongační faktor Tu MeSH
• guanosindifosfát MeSH
• guanosintrifosfát MeSH
• proteiny z Escherichia coli MeSH
• rekombinantní fúzní proteiny MeSH