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Direct Epoxidation in Candida antarctica Lipase B Studied by Experiment and Theory
KTH, Skolan för bioteknologi (BIO), Biokemi.ORCID-id: 0000-0003-2371-8755
KTH, Skolan för kemivetenskap (CHE), Kemi, Fysikalisk kemi.
KTH, Skolan för bioteknologi (BIO), Biokemi.
KTH, Skolan för kemivetenskap (CHE), Kemi, Fysikalisk kemi.
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2008 (Engelska)Ingår i: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 9, nr 15, s. 2443-2451Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Candida antarctica lipase B (CALB) is a promiscuous serine hydrolase that, besides its native function, catalyzes different side reactions, such as direct epoxidation. A single-point mutant of CALB demonstrated a direct epoxidation reaction mechanism for the epoxidation of alpha,beta-unsaturated aldehydes by hydrogen peroxide in aqueous and organic solution. Mutation of the catalytically active Ser105 to alanine made the previously assumed indirect epoxidation reaction mechanism impossible. Gibbs free energies, activation parameters, and substrate selectivities were determined both computationally and experimentally. The energetics and mechanism for the direct epoxidation in CALB Ser105Ala were investigated that the reaction proceeds through a two step-mechanism with formation of an oxyanionic intermediate. The active-site residue His224 functions as a general acid-base catalyst with support from Asp187. Oxyanion stabilization is facilitated by two hydrogen bonds from Thr40.

Ort, förlag, år, upplaga, sidor
2008. Vol. 9, nr 15, s. 2443-2451
Nyckelord [en]
catalytic promiscuity, enzyme catalysis, epoxidation, hydrolases, molecular dynamics, HYDROGEN-PEROXIDE, ACTIVE-SITE, PEROXYCARBOXYLIC ACIDS, EFFICIENT GENERATION, AM1-BCC MODEL, CATALYSIS, ALKENES, ENZYMES, OXIDATION, CHLOROPEROXIDASE
Identifikatorer
URN: urn:nbn:se:kth:diva-13270DOI: 10.1002/cbic.200800318ISI: 000260591100015Scopus ID: 2-s2.0-54349127157OAI: oai:DiVA.org:kth-13270DiVA, id: diva2:323008
Anmärkning
QC20100608Tillgänglig från: 2010-06-09 Skapad: 2010-06-09 Senast uppdaterad: 2017-12-12Bibliografiskt granskad
Ingår i avhandling
1. Lipase and ω-Transaminase: Biocatalytic Investigations
Öppna denna publikation i ny flik eller fönster >>Lipase and ω-Transaminase: Biocatalytic Investigations
2010 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Ort, förlag, år, upplaga, sidor
Stockholm: KTH, 2010. s. 67
Serie
Trita-BIO-Report, ISSN 1654-2312 ; 2010:10
Nyckelord
Candida antarctica lipase B, enzyme catalysis, enzyme catalytic promiscuity, molecular modeling, ω-transaminase
Nationell ämneskategori
Biokemi och molekylärbiologi
Identifikatorer
urn:nbn:se:kth:diva-13279 (URN)978-91-7415-648-5 (ISBN)
Disputation
2010-06-11, Svedbergssalen FD5, Roslagstullsbacken 21, AlbaNova, Stockholm, 10:00 (Engelska)
Opponent
Handledare
Anmärkning
QC20100609Tillgänglig från: 2010-06-09 Skapad: 2010-06-09 Senast uppdaterad: 2010-07-02Bibliografiskt granskad

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Svedendahl, MariaCarlqvist, PeterBranneby, CeciliaAllnér, OlofFrise, AntonHult, KarlBerglund, PerBrinck, Tore
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BiokemiFysikalisk kemi
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