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Exploring Conjugate Addition Activity in Pseudozyma antarctica Lipase B
KTH, School of Biotechnology (BIO). (Biochemistry)ORCID iD: 0000-0003-2371-8755
2009 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

Multifunctional enzymes have alternative functions or activities, known as “moonlighting” or “promiscuous”, which are often hidden behind a native enzyme activity and therefore only visible under special environmental conditions. In this thesis, the active-site of Pseudozyma (formerly Candida) antarctica lipase B was explored for a promiscuous conjugate addition activity. Pseudozyma antarctica lipase B is a lipase industrially used for hydrolysis or transacylation reactions. This enzyme contains a catalytic triad, Ser105-His224-Asp187, where a nucleophilic attack from Ser105 on carboxylic acid/ester substrates cause the formation of an acyl enzyme. For conjugate addition activity in Pseudozyma antarctica lipase B, replacement of Ser105 was assumed necessary to prevent competing hemiacetal formation. However, experiments revealed conjugate addition activity in both wild-type enzyme and the Ser105Ala variant. Enzyme-catalyzed conjugate additions were performed by adding sec-amine, thiols or 1,3-dicarbonyl compounds to various α,β-unsaturated carbonyl compounds in both water or organic solvent. The reactions followed Michaelis-Menten kinetics and the native ping pong bi bi reaction mechanism of Pseudozyma antarctica lipase B for hydrolysis/transacylation was rerouted to a novel ordered bi uni reaction mechanism for conjugate addition (Paper I, II, III). The lipase hydrolysis activity was suppressed more than 1000 times by the replacement of the nucleophilic Ser105 to Ala (Paper III).

Place, publisher, year, edition, pages
Stockholm: KTH , 2009. , 42 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2009:20
Series
TRITA-BIO-Report 2009:20, ISSN 1654-2312
Keyword [en]
Candida antarctica lipase B · conjugate addition · enzyme catalysis · enzyme catalytic promiscuity · molecular modeling
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-11598ISBN: 978-91-7415-435-1 (print)OAI: oai:DiVA.org:kth-11598DiVA: diva2:278042
Presentation
2009-10-16, M22, Brinellvägen 64, KTH, 10:00 (English)
Opponent
Supervisors
Available from: 2009-11-30 Created: 2009-11-23 Last updated: 2010-10-29Bibliographically approved
List of papers
1. Exploring the Active-Site of a Rationally Redesigned Lipase for Catalysis of Michael-Type Additions
Open this publication in new window or tab >>Exploring the Active-Site of a Rationally Redesigned Lipase for Catalysis of Michael-Type Additions
Show others...
2005 (English)In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 6, 331-336 p.Article in journal (Refereed) Published
Abstract [en]

Michael-type additions of various thiols and alpha,beta-unsaturated carbonyl compounds were performed in organic solvent catalyzed by wild-type and a rationally redesigned mutant of Candida antarctica lipase B. The mutant locks the nucleophilic serine 105 in the active-site; this results in a changed catalytic mechanism of the enzyme. The possibility of utilizing this mutant for Michael-type additions was initially explored by quantum-chemical calculations on the reaction between acrolein and methanethiol in a model system. The model system was constructed on the basis of docking and molecular-dynamics simulations and was designed to simulate the catalytic properties of the active site. The catalytic system was explored experimentally with a range of different substrates. The k(cat) values were found to be in the range of 10(-3) to 4 min(-1), similar to the values obtained with aldolase antibodies. The enzyme proficiency was 10(7). Furthermore, the Michael-type reactions followed saturation kinetics and were confirmed to take place in the enzyme active site.

Keyword
DENSITY-FUNCTIONAL THEORY; ENZYMATIC-REACTIONS; HYDROLYTIC ENZYMES; ALKALINE PROTEASE; BACILLUS-SUBTILIS; ORGANIC MEDIA; GROUND-STATE; MECHANISM
Identifiers
urn:nbn:se:kth:diva-5113 (URN)10.1002/cbic.200400213 (DOI)000226957100014 ()2-s2.0-20544452621 (Scopus ID)
Note
QC20100609Available from: 2005-05-15 Created: 2005-05-15 Last updated: 2017-12-04Bibliographically approved
2. Fast carbon-carbon bond formation by a promiscuous lipase
Open this publication in new window or tab >>Fast carbon-carbon bond formation by a promiscuous lipase
2005 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 127, no 51, 17988-17989 p.Article in journal (Refereed) Published
Keyword
ACTIVE TRIFLUOROMETHYLATED COMPOUNDS, CATALYZED MICHAEL ADDITION, ORGANIC MEDIA, HYDROLYTIC ENZYMES, 1, 3-DICARBONYL COMPOUNDS, CANDIDA-ANTARCTICA, ALKALINE PROTEASE, BACILLUS-SUBTILIS, BIOCATALYSIS, SITE
Identifiers
urn:nbn:se:kth:diva-13271 (URN)10.1021/ja056660r (DOI)000234258700012 ()2-s2.0-29844436474 (Scopus ID)
Note
QC20100609Available from: 2010-06-09 Created: 2010-06-09 Last updated: 2010-10-29Bibliographically approved
3. Suppressed Native Hydrolytic Activity of a Lipase to Reveal Promiscuous Michael Addition Activity in Water
Open this publication in new window or tab >>Suppressed Native Hydrolytic Activity of a Lipase to Reveal Promiscuous Michael Addition Activity in Water
2009 (English)In: CHEMCATCHEM, ISSN 1867-3880, Vol. 1, no 2, 252-258 p.Article in journal (Refereed) Published
Abstract [en]

Suppression of,the,native hydrolytic activity of Pseudozyma antarctica lipase B (PalB) (formerly Candida antarctica lipase B) in water is demonstrated. By replacing the catalytic Ser 105 residue with an alanine unit, promiscuous Michael addition activity is favored. A Michael addition reaction between methyl acrylate and acetylacetone was explored as a model system. For the PalB Ser 105 Ala mutant, the hydrolytic activity was suppressed more than 1000 times and at the same time, the Michael addition activity was increased by a factor of 100. Docking studies and molecular dynamics simulations revealed an increased ability of the PalB Ser 105 Ala mutant to harbor the substrates close to a catalytically competent conformation.

Keyword
enzyme catalysis, enzyme promiscuity, lipases, Michael addition, molecular modeling, CANDIDA-ANTARCTICA, PYRIMIDINE-DERIVATIVES, CATALYTIC PROMISCUITY, ALKALINE PROTEASE, BACILLUS-SUBTILIS, ORGANIC MEDIA, OLD ENZYMES, FORCE-FIELD, BIOCATALYSIS
Identifiers
urn:nbn:se:kth:diva-13269 (URN)10.1002/cctc.200900041 (DOI)000274153900009 ()2-s2.0-77958074145 (Scopus ID)
Note
QC20100609Available from: 2010-06-09 Created: 2010-06-09 Last updated: 2010-10-29Bibliographically approved

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