• MeSH
• enzymy chemie   genetika   metabolismus   MeSH
• koenzymy chemie   metabolismus   MeSH
• konformace proteinů MeSH
• proteinové inženýrství metody   MeSH
• rekombinantní proteiny chemie   genetika   metabolismus   MeSH
• rozpouštědla MeSH
• terciární struktura proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• enzymy MeSH
• koenzymy MeSH
• rekombinantní proteiny MeSH
• rozpouštědla MeSH

Engineering enzymes to degrade anthropogenic compounds efficiently is challenging. We obtained Rhodococcus rhodochrous haloalkane dehalogenase mutants with up to 32-fold higher activity than wild type toward the toxic, recalcitrant anthropogenic compound 1,2,3-trichloropropane (TCP) using a new strategy. We identified key residues in access tunnels connecting the buried active site with bulk solvent by rational design and randomized them by directed evolution. The most active mutant has large aromatic residues at two out of three randomized positions and two positions modified by site-directed mutagenesis. These changes apparently enhance activity with TCP by decreasing accessibility of the active site for water molecules, thereby promoting activated complex formation. Kinetic analyses confirmed that the mutations improved carbon-halogen bond cleavage and shifted the rate-limiting step to the release of products. Engineering access tunnels by combining computer-assisted protein design with directed evolution may be a valuable strategy for refining catalytic properties of enzymes with buried active sites.

• MeSH
• biodegradace MeSH
• cirkulární dichroismus MeSH
• hydrolasy chemie   genetika   metabolismus   MeSH
• látky znečišťující životní prostředí chemie   MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• mutageneze cílená MeSH
• počítačová simulace MeSH
• propan analogy a deriváty   chemie   MeSH
• proteinové inženýrství * MeSH
• Rhodococcus enzymologie   genetika   růst a vývoj   MeSH
• řízená evoluce molekul MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 1,2,3-trichloropropane MeSH Prohlížeč
• haloalkane dehalogenase MeSH Prohlížeč
• hydrolasy MeSH
• látky znečišťující životní prostředí MeSH
• propan MeSH

HotSpot Wizard is a web server for automatic identification of 'hot spots' for engineering of substrate specificity, activity or enantioselectivity of enzymes and for annotation of protein structures. The web server implements the protein engineering protocol, which targets evolutionarily variable amino acid positions located in the active site or lining the access tunnels. The 'hot spots' for mutagenesis are selected through the integration of structural, functional and evolutionary information obtained from: (i) the databases RCSB PDB, UniProt, PDBSWS, Catalytic Site Atlas and nr NCBI and (ii) the tools CASTp, CAVER, BLAST, CD-HIT, MUSCLE and Rate4Site. The protein structure and e-mail address are the only obligatory inputs for the calculation. In the output, HotSpot Wizard lists annotated residues ordered by estimated mutability. The results of the analysis are mapped on the enzyme structure and visualized in the web browser using Jmol. The HotSpot Wizard server should be useful for protein engineers interested in exploring the structure of their favourite protein and for the design of mutations in site-directed mutagenesis and focused directed evolution experiments. HotSpot Wizard is available at http://loschmidt.chemi.muni.cz/hotspotwizard/.

• MeSH
• beta-laktamasy chemie   MeSH
• glykosidhydrolasy chemie   MeSH
• hydrolasy triesterů kyseliny fosforečné chemie   MeSH
• hydrolasy chemie   MeSH
• internet MeSH
• proteinové inženýrství * MeSH
• reprodukovatelnost výsledků MeSH
• software * MeSH
• uživatelské rozhraní počítače MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• beta-laktamasy MeSH
• glykosidhydrolasy MeSH
• haloalkane dehalogenase MeSH Prohlížeč
• hydrolasy triesterů kyseliny fosforečné MeSH
• hydrolasy MeSH
• licheninase MeSH Prohlížeč