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Fast carbon-carbon bond formation by a promiscuous lipase
KTH, School of Biotechnology (BIO), Biochemistry.ORCID iD: 0000-0003-2371-8755
KTH, School of Biotechnology (BIO), Biochemistry.
KTH, School of Biotechnology (BIO), Biochemistry.ORCID iD: 0000-0002-9577-832X
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
Place, publisher, year, edition, pages
2005. Vol. 127, no 51, 17988-17989 p.
Keyword [en]
ACTIVE TRIFLUOROMETHYLATED COMPOUNDS, CATALYZED MICHAEL ADDITION, ORGANIC MEDIA, HYDROLYTIC ENZYMES, 1, 3-DICARBONYL COMPOUNDS, CANDIDA-ANTARCTICA, ALKALINE PROTEASE, BACILLUS-SUBTILIS, BIOCATALYSIS, SITE
Identifiers
URN: urn:nbn:se:kth:diva-13271DOI: 10.1021/ja056660rISI: 000234258700012Scopus ID: 2-s2.0-29844436474OAI: oai:DiVA.org:kth-13271DiVA: diva2:323001
Note
QC20100609Available from: 2010-06-09 Created: 2010-06-09 Last updated: 2017-12-12Bibliographically approved
In thesis
1. Lipase and ω-Transaminase: Biocatalytic Investigations
Open this publication in new window or tab >>Lipase and ω-Transaminase: Biocatalytic Investigations
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In a lipase investigation, Candida antarctica lipase B (CALB) are explored for enzyme catalytic promiscuity. Enzyme catalytic promiscuity is shown by enzymes catalyzing alternative catalytic transformations proceeding via different transition state structures than normal. CALB normally performs hydrolysis reactions by activating and coordinating carboxylic acid/ester substrates in an oxyanion hole prior to nucleophilic attack from an active-site serine resulting in acyl enzyme formation. The idea of utilizing the carbonyl activation oxyanion hole in the active-site of CALB to catalyze promiscuous reactions arose by combining catalytic and structural knowledge about the enzyme with chemical imagination. We choose to explore conjugate addition and direct epoxidation activities in CALB by combining molecular modeling and kinetic experiments. By quantum-chemical calculations, the investigated promiscuous reactions were shown to proceed via ordered reaction mechanisms that differ from the native ping pong bi bi reaction mechanism. The investigated promiscuous activities were shown to take place in the enzyme active-site by various kinetic experiments, but despite this, no enantioselectivity was displayed. The reason for this is unknown, but can be a result of a too voluminous active-site or the lack of covalent coordination of the substrates during enzyme-catalysis (Paper I-IV). Combining enzyme structural knowledge with chemical imagination may provide numerous novel enzyme activities to be discovered. In an ω-transaminase investigation, two (S)-selective ω-transaminases from Arthrobacter citreus (Ac-ωTA) and Chromobacterium violaceum (Cv-ωTA) are explored aiming to improve their catalytic properties. Structural knowledge of these enzymes was provided by homology modeling. A homology structure of Ac-ωTA was successfully applied for rational design resulting in enzyme variants with improved enantioselectivity. Additionally, a single-point mutation reversed the enantiopreference of the enzyme from (S) to (R), which was further shown to be substrate dependent (Paper V). A homology structure of Cv-ωTA guided the creation of an enzyme variant showing reduced isopropyl amine inhibition.

Place, publisher, year, edition, pages
Stockholm: KTH, 2010. 67 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2010:10
Keyword
Candida antarctica lipase B, enzyme catalysis, enzyme catalytic promiscuity, molecular modeling, ω-transaminase
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-13279 (URN)978-91-7415-648-5 (ISBN)
Public defence
2010-06-11, Svedbergssalen FD5, Roslagstullsbacken 21, AlbaNova, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
QC20100609Available from: 2010-06-09 Created: 2010-06-09 Last updated: 2010-07-02Bibliographically approved
2. Exploring Conjugate Addition Activity in Pseudozyma antarctica Lipase B
Open this publication in new window or tab >>Exploring Conjugate Addition Activity in Pseudozyma antarctica Lipase B
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
Candida antarctica lipase B · conjugate addition · enzyme catalysis · enzyme catalytic promiscuity · molecular modeling
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-11598 (URN)978-91-7415-435-1 (ISBN)
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

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Svedendahl, MariaBerglund, Per

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