enzyme engineering
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Annals of the New York Academy of Sciences, ISSN 0077-8923 Volume 542, Issue 1, December 1988
[1st ed.] XI, 535 s. : tab., grafy ; 22 cm
- MeSH
- biologické přípravky biosyntéza MeSH
- biotechnologie přístrojové vybavení metody MeSH
- enzymy fyziologie MeSH
- Publikační typ
- kongresy MeSH
- sborníky MeSH
- Konspekt
- Biochemie. Molekulární biologie. Biofyzika
- NLK Obory
- biochemie
Annals of the New York Academy of Sciences ; Vol. 613, no. 1, 1990
[1st ed.] XXII, 897 s. : obr., tab., grafy ; 23 cm
Annals of the New York Academy of Sciences ; vol. 501, no. 1, 1987
573 s. : obr., tab. ; 23 cm
Enzymes are efficient and specific catalysts for many essential reactions in biotechnological and pharmaceutical industries. Many times, the natural enzymes do not display the catalytic efficiency, stability or specificity required for these industrial processes. The current enzyme engineering methods offer solutions to this problem, but they mainly target the buried active site where the chemical reaction takes place. Despite being many times ignored, the tunnels and channels connecting the environment with the active site are equally important for the catalytic properties of enzymes. Changes in the enzymatic tunnels and channels affect enzyme activity, specificity, promiscuity, enantioselectivity and stability. This review provides an overview of the emerging field of enzyme access tunnel engineering with case studies describing design of all the aforementioned properties. The software tools for the analysis of geometry and function of the enzymatic tunnels and channels and for the rational design of tunnel modifications will also be discussed. The combination of new software tools and enzyme engineering strategies will provide enzymes with access tunnels and channels specifically tailored for individual industrial processes.
Protein engineering strategies aimed at constructing enzymes with novel or improved activities, specificities, and stabilities greatly benefit from in silico methods. Computational methods can be principally grouped into three main categories: bioinformatics; molecular modelling; and de novo design. Particularly de novo protein design is experiencing rapid development, resulting in more robust and reliable predictions. A recent trend in the field is to combine several computational approaches in an interactive manner and to complement them with structural analysis and directed evolution. A detailed investigation of designed catalysts provides valuable information on the structural basis of molecular recognition, biochemical catalysis, and natural protein evolution.
- MeSH
- enzymy genetika MeSH
- lidé MeSH
- molekulární modely MeSH
- mutace MeSH
- proteinové inženýrství metody MeSH
- stabilita enzymů MeSH
- výpočetní biologie metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
- MeSH
- biomedicínské inženýrství MeSH
- enzymy imobilizované MeSH
- katalýza MeSH
- kinetika MeSH
- transportní proteiny MeSH
- Publikační typ
- přehledy MeSH
Fabryho choroba je na X-chromozóm viazané dedičné metabolické ochorenie, charakterizované defektom odbúravania glykosfingolipidov. Tieto sa v dôsledku ochorenia hromadia v rôznych tkanivách vo forme hustých depozitov. Základnou príčinou ochorenia je mutácia génu lokalizovaného na dlhom ramienku X-chromozómu, kódujúceho lyzozomálny enzým alfa-galaktozidáza A. Fabryho choroba (FCH) je tiež známa ako Andersonova-Fabryho choroba alebo angiokeratoma corporis diffusum. Patrí medzi zriedkavo sa vyskytujúce ochorenia, označované aj ako „orphan diseases“. FCH je progresívne ochorenie a jej prognóza bez liečby je zlá. Pokroky vo farmaceutickom priemysle ponúkajú nové terapeutické možnosti v paliatívnej (symtomatickej) liečbe, vedúce k miernemu zlepšeniu prognózy. Priemerná očakávaná dĺžka života u postihnutých mužov je 50–60 rokov. Postihnuté ženy sa dožívajú 60–70 rokov. Ich prognóza je vo všeobecnosti lepšia ako u mužov, u ktorých sa v neskorších rokoch života prejavujú závažné príznaky ochorenia. Najčastejšou príčinou úmrtia sú kardiovaskulárne príhody a renálne zlyhania. K významnému prelomu došlo v roku 1989, keď Kornreich so spolupracovníkmi rozlúštili genetický kód enzýmu alfa-galaktozidázy, čo umožnilo jeho laboratórnu prípravu. Enzýmová substitučná terapia (EST) prináša zlepšenie kvality života pre pacientov s FCH a niektorých z ďalších zriedkavo sa vyskytujúcich ochorení. Pre substitučnú liečbu sú dostupné dva komerčné prípravky alfa-galaktozidázy A. Oba sú pripravované umelo, z geneticky upravených buniek: z kultivovaných ľudských fibroblastov v prípade agalzidázy-alfa (REPLAGALTM) a z ovariálnych buniek čínskych škrečkov pri agalzidáze-beta (FABRAZYME®).
Fabry disease is an X–linked, hereditary metabolic disorder characterized by a defect in the degradation of glycosphingolipids. It leads to their accumulation as lysosomal dense bodies in various tissues. The underlying cause is mutation in the gene located on the long arm of the X chromosome encoding the lysosomal enzyme, α-galactosidase A. Fabry disease (FD) is also known as Anderson-Fabry disease or angiokeratoma corporis diffusum. It belongs to rare disorders, so-called „orphan diseases“. FD takes a progressive course and the prognosis, if untreated, is bleak. Advances made in the pharmaceutical industry have offered new therapeutic possibilities in palliative (symptomatic) treatment, leading to a slight improvement in prognosis. The average life expectancy of affected males is 50–60 years. Female patients survive to their sixth or seventh decade of life. Their prognosis is generally better than in male patients with more serious manifestations of the disease occurring later in life. The main causes of death are cardiovascular events and renal failure. A major breakthrough came in 1989, when Kornreich and co-workers deciphered the genetic code of α-galactosidase, enabling the enzyme to be produced laboratory. Enzyme replacement therapy (ERT) has brought improving quality of life for the patients with FD and some of the others orphan diseases. Two commercial products of α-galactosidase A are available for ERT. Both are produced artificially from genetically engineered cells: cultured human fibroblasts in case of agasidase-alfa (REPLAGALTM); and Chinese hamster ovary cells in the case of agalsidase-beta (FABRAZYME®)
- Klíčová slova
- Replagal, Fabrazyme,
- MeSH
- Evropská unie MeSH
- Fabryho nemoc etiologie farmakoterapie terapie MeSH
- genetické inženýrství metody využití MeSH
- lidé MeSH
- lyzozomální střádavé nemoci v nervovém systému farmakoterapie MeSH
- prognóza MeSH
- výroba orphan drugs ekonomika zákonodárství a právo MeSH
- Check Tag
- lidé MeSH
The transplantation of loops between structurally related proteins is a compelling method to improve the activity, specificity and stability of enzymes. However, despite the interest of loop regions in protein engineering, the available methods of loop-based rational protein design are scarce. One particular difficulty related to loop engineering is the unique dynamism that enables them to exert allosteric control over the catalytic function of enzymes. Thus, when engaging in a transplantation effort, such dynamics in the context of protein structure need consideration. A second practical challenge is identifying successful excision points for the transplantation or grafting. Here, we present LoopGrafter (https://loschmidt.chemi.muni.cz/loopgrafter/), a web server that specifically guides in the loop grafting process between structurally related proteins. The server provides a step-by-step interactive procedure in which the user can successively identify loops in the two input proteins, calculate their geometries, assess their similarities and dynamics, and select a number of loops to be transplanted. All possible different chimeric proteins derived from any existing recombination point are calculated, and 3D models for each of them are constructed and energetically evaluated. The obtained results can be interactively visualized in a user-friendly graphical interface and downloaded for detailed structural analyses.
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
- práce podpořená grantem MeSH
The gene encoding an epoxide hydrolase from Aspergillus niger M200 has been cloned and its sequence determined. The gene is interrupted by seven introns, one exon being only nine nucleotides long. The non-coding 5'- and 3'-regions of the mRNA are composed of 47 and 76 nucleotides, respectively. Overexpression of the fungal epoxide hydrolase in E. coli TOP10 has led to a 15-fold increase in specific activity (compared to the wild-type strain). Saturation mutagenesis at codon 217 resulted in the discovery of nine enzyme variants showing in several cases profound differences in activity and enantioselectivity towards various epoxides when compared to the data of the wild-type enzyme. The site 217 is located at the entrance of the tunnel that provides the substrate with access to the active site. The exchange of Ala at this position for Cys has led to a doubled enantioselectivity (E-value of 5.0) towards benzyl glycidyl ether. The same substitution resulted in a threefold-enhanced activity of the enzyme towards allyl glycidyl ether and styrene oxide without affecting enantioselectivity. The variant A217L showed an enhanced enantioselectivity towards tert-butyl glycidyl ether reaching an E-value of 100 (from 60 for the wild-type enzyme). Replacement of A217 by Val has led to higher activity towards allyl glycidyl ether by a factor of six. The substitutions Ala-->Glu and Ala-->Gln increased the enantioselectivity towards allyl glycidyl ether and styrene oxide by over 50% to E-values of 10 and 16, respectively. The study underlines that single amino acid exchanges in the substrate tunnel region can lead to significant improvements in enantioselectivity and activity of the epoxide hydrolase from A. niger M200.
- MeSH
- Aspergillus niger enzymologie genetika MeSH
- biotechnologie MeSH
- DNA fungální genetika MeSH
- epoxid hydrolasy genetika chemie metabolismus MeSH
- Escherichia coli genetika MeSH
- financování organizované MeSH
- fylogeneze MeSH
- geny hub MeSH
- katalytická doména MeSH
- kinetika MeSH
- klonování DNA MeSH
- komplementární DNA genetika MeSH
- molekulární modely MeSH
- mutageneze cílená MeSH
- proteinové inženýrství MeSH
- rekombinantní proteiny genetika chemie metabolismus MeSH
- sekvence nukleotidů MeSH
- stereoizomerie MeSH
- substituce aminokyselin MeSH
- substrátová specifita MeSH
