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Engineering carbonic anhydrase for highly selective ester hydrolysis
Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology . Linköping University, The Institute of Technology.ORCID iD: 0000-0003-1032-2145
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 [en]
carbonic anhydrase, specificity, hydrolysis, rational design, protein engineering, plasmodium falciparum
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:liu:diva-10477ISBN: 978-91-85715-43-5 (print)OAI: oai:DiVA.org:liu-10477DiVA: diva2:17229
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
List of papers
1. Redesign of human carbonic anhydrase II for increased esterase activity and specificity towards esters with long acyl chains
Open this publication in new window or tab >>Redesign of human carbonic anhydrase II for increased esterase activity and specificity towards esters with long acyl chains
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.

Keyword
Carbonic anhydrase, Hydrolysis, Specificity, Mutagenesis, Protein engineering, Rational design
National Category
Engineering and Technology
Identifiers
urn:nbn:se:liu:diva-12859 (URN)10.1016/j.bbapap.2006.07.010 (DOI)
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
2. Converting human carbonic anhydrase II into a benzoate ester hydrolase through rational redesign
Open this publication in new window or tab >>Converting human carbonic anhydrase II into a benzoate ester hydrolase through rational redesign
2008 (English)In: Biochimica et Biophysica Acta, ISSN 0006-3002, Vol. 1784, no 5, 811-815 p.Article in journal (Refereed) Published
Abstract [en]

Enzymes capable of benzoate ester hydrolysis have several potential medical and industrial applications. A variant of human carbonic anhydrase II (HCAII) was constructed, by rational design, that is capable of hydrolysing para-nitrophenyl benzoate (pNPBenzo) with an efficiency comparable to some naturally occuring esterases. The design was based on a previously developed strategy,[1] in which docking of a transition state analogue (TSA) to the active site of HCAII was used to predict mutations that would allow the reaction. A triple mutant, V121A/V143A/T200A, was thus constructed and shown to hydrolyze pNPBenzo with kcat/KM = 625 (± 38) M-1s-1. It is highly active with other ester substrates as well, and hydrolyzes para-nitrophenyl acetate with kcat/KM = 101700 (± 4800) M-1s-1, which is the highest esterase efficiency so far for any CA variant. A parent mutant (V121A/V143A) has measurable KM values for para-nitrophenyl butyrate (pNPB) and valerate (pNPV),[1] but for V121A/V143A/T200A no KM could be determined, showing that the additional T200A mutation has caused a decreased substrate binding. However, kcat/KM is higher with both substrates for the triple mutant, indicating that binding energy has been diverted from substrate binding to transition state stabilization.

Place, publisher, year, edition, pages
Institutionen för fysik, kemi och biologi, 2008
Keyword
Carbonic anhydrase, Hydrolysis, Mutagenesis, Protein engineering, Rational design, Specificity
National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-11787 (URN)10.1016/j.bbapap.2008.02.007 (DOI)
Note
Original publication: Gunnar E. Höst and Bengt-Harald Jonsson, Converting human carbonic anhydrase II into a benzoate ester hydrolase through rational redesign, 2008, Biochimica et Biophysica Acta, (1784), 5, 811-815. http://dx.doi.org/10.1016/j.bbapap.2008.02.007. Copyright: Elsevier B.V., http://www.elsevier.com/Available from: 2008-05-13 Created: 2008-05-13 Last updated: 2013-09-12
3. Combined Enzyme and Substrate Design: Grafting of a Cooperative Two-Histidine Catalytic Motif into a Protein Targeted at the Scissile Bond in a Designed Ester Substrate
Open this publication in new window or tab >>Combined Enzyme and Substrate Design: Grafting of a Cooperative Two-Histidine Catalytic Motif into a Protein Targeted at the Scissile Bond in a Designed Ester Substrate
2007 (English)In: ChemBioChem (Print), ISSN 1439-4227, Vol. 8, no 13, 1570-1576 p.Article in journal (Refereed) Published
Abstract [en]

A histidine-based, two-residue reactive site for the catalysis of hydrolysis of designed sulfonamide-containing para-nitrophenyl esters has been engineered into a scaffold protein. A matching substrate was designed to exploit the natural active site of human carbonic anhydrase II (HCAII) for well-defined binding. In this we took advantage of the high affinity between the active site zinc atom and sulfonamides. The ester substrate was designed to position the scissile bond in close proximity to the His64 residue in the scaffold protein. Three potential sites for grafting the catalytic His-His pair were identified, and the corresponding N62H/H64, F131H/V135H and L198H/P202H mutants were constructed. The most efficient variant, F131H/V135H, has a maximum kcat/KM value of approximately 14 000 M-1 s-1, with a kcat value that is increased by a factor of 3 relative to that of the wild-type HCAII, and by a factor of over 13 relative to the H64A mutant. The results show that an esterase can be designed in a stepwise way by a combination of substrate design and grafting of a designed catalytic motif into a well-defined substrate binding site.

Keyword
chiral resolution, enzyme catalysis, hydrolysis, mutagenesis, protein engineering
National Category
Engineering and Technology
Identifiers
urn:nbn:se:liu:diva-12861 (URN)10.1002/cbic.200600540 (DOI)
Available from: 2008-01-09 Created: 2008-01-09 Last updated: 2013-09-12
4. Cloning of a putative carbonic anhydrase from the human malaria parasite Plasmodium falciparum
Open this publication in new window or tab >>Cloning of a putative carbonic anhydrase from the human malaria parasite Plasmodium falciparum
(English)Manuscript (Other academic)
Identifiers
urn:nbn:se:liu:diva-12862 (URN)
Available from: 2008-01-09 Created: 2008-01-09 Last updated: 2013-09-12

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