liu.seSearch for publications in DiVA
Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • oxford
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Redesign of human carbonic anhydrase II for increased esterase activity and specificity towards esters with long acyl chains
Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.ORCID iD: 0000-0003-1032-2145
Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.ORCID iD: 0000-0002-7642-9263
Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.
2006 (English)In: Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics, ISSN 1570-9639, Vol. 1764, no 10, 1601-1606 p.Article in journal (Refereed) Published
Abstract [en]

The effect of modulating the shape and the size of the hydrophobic pocket on the esterase activity and specificity of human carbonic anhydrase II (HCAII) for esters with different acyl chain lengths was investigated. Following an initial screen of 7 HCAII variants with alanine substitutions in positions 121, 143 and 198, detailed kinetic measurements were performed on HCAII and the variants V121A, V143A and V121A/V143A. For some variants, an increased size of the hydrophobic pocket resulted in increased activities and specificities for longer substrates. For V121A/V143A, the rate of hydrolysis for paranitrophenyl valerate was increased by a factor of approximately 3000. The specificities also changed dramatically, for example V121A/V143A is 6.3 times more efficient with paranitrophenyl valerate than paranitrophenyl acetate, while HCAII is > 500 times more efficient with paranitrophenyl acetate than paranitrophenyl valerate. An automated docking procedure was performed on these variants with transition state analogues (TSAs) for the hydrolysis reaction. It was possible to correlate the catalytic rate constants to the docking results, i.e. for each variant, efficient hydrolysis was generally correlated to successful TSA-docking. The observations in this paper show that the redesign increased the catalytic rates for substrates with long acyl chains by removal of steric hinders and addition of new favourable binding interactions.

Place, publisher, year, edition, pages
2006. Vol. 1764, no 10, 1601-1606 p.
Keyword [en]
Carbonic anhydrase, Hydrolysis, Specificity, Mutagenesis, Protein engineering, Rational design
National Category
Engineering and Technology
Identifiers
URN: urn:nbn:se:liu:diva-12859DOI: 10.1016/j.bbapap.2006.07.010OAI: oai:DiVA.org:liu-12859DiVA: diva2:17225
Note
Original Publication: Gunnar Höst, Lars-Göran Mårtensson and Bengt-Harald Jonsson, Redesign of human carbonic anhydrase II for increased esterase activity and specificity towards esters with long acyl chains, 2006, Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics, (1764), 10, 1601-1606. http://dx.doi.org/10.1016/j.bbapap.2006.07.010 Copyright: Elsevier http://www.elsevier.com/ Available from: 2008-01-09 Created: 2008-01-09 Last updated: 2013-10-04
In thesis
1. Engineering carbonic anhydrase for highly selective ester hydrolysis
Open this publication in new window or tab >>Engineering carbonic anhydrase for highly selective ester hydrolysis
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [sv]

I denna avhandling presenteras arbete utfört med enzymet humant karboanhydras II (HCAII). Enzymer är en typ av proteiner som accelererar (katalyserar) kemiska reaktioner, vilket är nödvändigt för allt levande. Den naturliga funktionen för HCAII är att katalysera omvandlingen av gasen koldioxid till vätekarbonat, som är löslig i vätska. Detta är viktigt bl.a. för att koldioxid som bildas i kroppen, och fraktas i blodet i form av vätekarbonat, skall hinna över till utandningsluften under den korta tid blodet är i lungorna.

Proteiner består av aminosyror som länkats samman i en lång kedja, där varje aminosyra är en av de 20 naturliga aminosyratyperna. Ett proteins struktur och egenskaper bestäms av aminosyrasekvensen, som i sin tur bestäms av genen för just det proteinet. Med genteknik kan ett proteins gen ändras (muteras), så att aminosyrasekvensen ändras, och det har här utnyttjats för att förändra HCAIIs katalytiska egenskaper. Förutom dess naturliga funktion kan HCAII även klyva (hydrolysera) vissa estrar. Mutationer gjordes så att en ’ficka’ i HCAIIs struktur, där molekylerna (substraten) som skall klyvas binder, fick en större volym. På så sätt skapades varianter med en kraftigt ökad kapacitet för att hydrolysera långa estersubstrat jämfört med icke-muterat HCAII. Som en utveckling av detta projekt skapades en mutant av HCAII, som kan hydrolysera ett än mer skrymmande substrat.

I ett annat projekt har en ny katalytisk aktivitet skapats i HCAII, som inte utnyttjar enzymets naturliga katalytiska förmåga. Ett nytt estersubstrat konstruerades, med en del som binder kraftigt till HCAII, så att en stark substratbindning erhölls. Sedan muterades vissa aminosyror till en reaktiv aminosyra som heter histidin. Valet av positioner för mutation baserades på en datormodell av enzymet med bundet substrat. Eftersom histidin kan delta i hydrolysreaktioner, får det muterade enzymet möjlighet att klyva substratet. Flera olika mutanter testades, och den effektivaste innehöll ett nära kopplat par av histidiner. Denna mutant undersöktes mer noggrannt, vilket gav viss information om den katalytiska mekanismen.

Det långsiktiga målet med detta arbete är att konstruera muterade enzymer som kan klyva giftiga ämnen, eller användas vid framställning av kemikalier. Det finns behov av nya enzymer för olika typer av substrat, och att med rationella metoder skapa nya katalytiska aktiviteter i proteiner är ett svårt vetenskapligt problem som ännu är i ett tidigt utvecklingsskede.

Abstract [en]

The main part of this thesis describes results from protein engineering experiments, in which the catalytic activity of the enzyme human carbonic anhydrase II (HCAII) is engineered by mutagenesis. This enzyme, which catalyzes the interconversion between CO2 and HCO3- in the body, also has the ability to hydrolyze ester bonds. In one project, the specificity of HCAII towards a panel of para-nitrophenyl ester substrates, with acyl chain lengths ranging from one to five carbon atoms, was changed by enlarging the substrate binding hydrophobic pocket. A variant was identified that has highly increased specificity towards substrates with long acyl chains. The mutant V121A/V143A hydrolyzes pNPV, which has four carbon atoms in the acyl chain, with an efficiency that is increased by a factor of 3000 compared to HCAII. Further, transition state analogues (TSAs) were docked to HCAII and mutant variants, and the results were correlated to the results from kinetic measurements. This indicated that automated docking could be used to some extent to construct HCAII variants with a designed specificity. Using this approach, a HCAII mutant that can hydrolyze a model benzoate ester was created. Interestingly, the resulting variant V121A/V143A/T200A was found to be highly active with other ester substrates as well. For pNPA, a kcat/KM of 1*105 M-1s-1 was achieved, which is the highest efficiency for hydrolysis of carboxylic acid esters reported for any HCAII variant.

In another project, the strong affinity between the active site zinc ion and sulfonamide was used to achieve binding of a designed substrate. Thus, the natural Zn-OH- site of HCAII was not used for catalysis, but for substrate binding. The substrate contains a benzenesulfonamide part in one end, with a para-nitrophenyl ester connected via a linker. The linker was chosen to ensure that the scissile bond is positioned close to His-64 and histidine residues introduced by mutagenesis in other positions. Using this approach, an enzyme was designed with a distinctly new two-histidine catalytic site for ester hydrolysis. The mutant, F131H/V135H, has a kcat/KM of approximately 14000 M-1s-1, which corresponds to a rate enhancement of 107 compared to a histidine mimic.

Finally, results are reported on a project aimed at cloning and producing a putative carbonic anhydrase from the malaria parasite Plasmodium falciparum. The gene was cloned by PCR and the construct was overexpressed in E. coli. However, the resulting protein was not soluble, and initial attempts to refold it are also reported.

Place, publisher, year, edition, pages
Institutionen för fysik, kemi och biologi, 2007
Series
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1085
Keyword
carbonic anhydrase, specificity, hydrolysis, rational design, protein engineering, plasmodium falciparum
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:liu:diva-10477 (URN)978-91-85715-43-5 (ISBN)
Public defence
2007-04-04, Planck, Fysikhuset, Campus Valla, Linköpings Universitet, Linköping, 10:15 (English)
Opponent
Supervisors
Available from: 2008-01-09 Created: 2008-01-09 Last updated: 2013-10-04

Open Access in DiVA

fulltext(385 kB)1813 downloads
File information
File name FULLTEXT01.pdfFile size 385 kBChecksum SHA-512
2506de34827d1eea1ca037347fbeccd38c3828eace29df3c613f5ef0ce1d8c0b43b89981c8cce7f47b5880cc7fbee35311aca20cd3363ca248ca92f459807624
Type fulltextMimetype application/pdf
attachment(261 kB)255 downloads
File information
File name ATTACHMENT01.pdfFile size 261 kBChecksum SHA-512
0dc28c36ee89f41619f454f117a41c870d56f0836332e1472c560af26a18dc6a7aac26b9f3f93df60e4d788a8216c8710e85b8ad218dde23e65b7786f4f89901
Type attachmentMimetype application/pdf

Other links

Publisher's full textLink to Ph.D. thesis

Authority records BETA

Höst, GunnarMårtensson, Lars-GöranJonsson, Bengt-Harald

Search in DiVA

By author/editor
Höst, GunnarMårtensson, Lars-GöranJonsson, Bengt-Harald
By organisation
Molecular Biotechnology The Institute of TechnologyBiochemistry
Engineering and Technology

Search outside of DiVA

GoogleGoogle Scholar
Total: 1813 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

doi
urn-nbn

Altmetric score

doi
urn-nbn
Total: 338 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • oxford
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf