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Quantum chemical studies of epoxide-transforming enzymes
KTH, School of Biotechnology (BIO).
2007 (English)Licentiate thesis, comprehensive summary (Other scientific)
Abstract [en]

Density functional theory is employed to study the reaction mechanisms of different epoxide-transforming enzymes. Calculations are based on quantum chemical active site models, which are build from X-ray crystal structures. The models are used to study conversion of various epoxides into their corresponding diols or substituted alcohols. Epoxide-transforming enzymes from three different families are studied. The human soluble epoxide hydrolase (sEH) belongs to the α/β-hydrolase fold family. sEH employs a covalent mechanism to hydrolyze various epoxides into vicinal diols. The Rhodococcus erythrobacter limonene epoxide hydrolase (LEH) constitutes a novel epoxide hydrolase, which is considered the founding member of a new family of enzymes. LEH mediates transformation of limone-1,2-epoxide into the corresponding vicinal diol by employing a general acid/general base-mediated mechanism. The Agrobacterium radiobacter AD1 haloalcohol dehalogenase HheC is related to the short-chain dehydrogenase/reductases. HheC is able to convert epoxides using various nucleophiles such as azide, cyanide, and nitrite. Reaction mechanisms of these three enzymes are analyzed in depth and the role of different active site residues is studied through in silico mutations. Steric and electronic factors influencing the regioselectivity of epoxide opening are identified. The computed energetics help to explain preferred reaction pathways and experimentally observed regioselectivities. Our results confirm the usefulness of the employed computational methodology for investigating enzymatic reactions.

Place, publisher, year, edition, pages
Stockholm: KTH , 2007. , x, 58 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2007:3
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-4390ISBN: 978-91-7178-640-1 (print)OAI: oai:DiVA.org:kth-4390DiVA: diva2:12099
Presentation
2007-05-11, FD41, AlbaNova, 10:00
Opponent
Supervisors
Note
QC 20101108Available from: 2007-05-22 Created: 2007-05-22 Last updated: 2010-11-08Bibliographically approved
List of papers
1. Catalytic mechanism of limonene epoxide hydrolase: a theoretical study
Open this publication in new window or tab >>Catalytic mechanism of limonene epoxide hydrolase: a theoretical study
2005 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 127, no 41, 14339-14347 p.Article in journal (Refereed) Published
Abstract [en]

The catalytic mechanism of limonene epoxide hydrolase (LEH) was investigated theoretically using the density functional theory method B3LYP. LEH is part of a novel limonene degradation pathway found in Rhodococcus erythropolis DCL14, where it catalyzes the hydrolysis of limonene-1,2-epoxide to give limonene-1,2-diol. The recent crystal structure of LEH was used to build a model of the LEH active site composed of five amino acids and a crystallographically observed water molecule. With this model, hydrolysis of different substrates was investigated. It is concluded that LEH employs a concerted general acid/general base-catalyzed reaction mechanism involving protonation of the substrate by Asp101, nucleophilic attack by water on the epoxide, and abstraction of a proton from water by Asp132. Furthermore, we provide an explanation for the experimentally observed regioselective hydrolysis of the four stereoisomers of limonene-1,2-epoxide.

Keyword
Amino acids; Catalysis; Degradation; Hydrolysis; Mathematical models; Probability density function; Limonene degradation; Limonene epoxide hydrolase (LEH); Regioselective hydrolysis; Rhodococcus erythropolis DCL14
National Category
Chemical Sciences
Identifiers
urn:nbn:se:kth:diva-7168 (URN)10.1021/ja050940p (DOI)000232605600057 ()2-s2.0-26844530203 (Scopus ID)
Note
QC 20100811Available from: 2007-05-22 Created: 2007-05-22 Last updated: 2010-08-11Bibliographically approved
2. Theoretical study of the full reaction mechanism of human soluble epoxide hydrolase
Open this publication in new window or tab >>Theoretical study of the full reaction mechanism of human soluble epoxide hydrolase
2006 (English)In: Chemistry - A European Journal, ISSN 0947-6539, E-ISSN 1521-3765, Vol. 12, no 26, 6898-6909 p.Article in journal (Refereed) Published
Abstract [en]

The complete reaction mechanism of soluble epoxide hydrolase (sEH) has been investigated by using the B3LYP density functional theory method. Epoxide hydrolases catalyze the conversion of epoxides to their corresponding vicinal diols. In our theoretical study, the sEH active site is represented by quantum-chemical models that are based on the X-ray crystal structure of human soluble epoxide hydrolase. The trans-substituted epoxide (1S,2S)-beta-methyl styrene oxide has been used as a substrate in the theoretical investigation of the sEH reaction mechanism. Both the alkylation and the hydrolytic half-reactions have been studied in detail. We present the energetics of the reaction mechanism as well as the optimized intermediates and transition-state structures. Full potential energy curves for the reactions involving nucleophilic attack at either the benzylic or the homo-benzylic carbon atom of (1S,2S)-beta-methylstyrene oxide have been computed. The regioselectivity of epoxide opening has been addressed for the two substrates (1S,2S)-beta-methylstyrene oxide and (S)-styrene oxide.

Keyword
density functional calculations; enzyme catalysis; epoxide hydrolase; reaction mechanisms; regioselectivity
National Category
Chemical Sciences
Identifiers
urn:nbn:se:kth:diva-7169 (URN)10.1002/chem.200501519 (DOI)000240387000017 ()2-s2.0-33748532821 (Scopus ID)
Note
QC 20100811Available from: 2007-05-22 Created: 2007-05-22 Last updated: 2010-08-11Bibliographically approved
3. Insights into the Reaction Mechanism of Soluble Epoxide Hydrolase from Theoretical Active Site Mutants
Open this publication in new window or tab >>Insights into the Reaction Mechanism of Soluble Epoxide Hydrolase from Theoretical Active Site Mutants
2006 (English)In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 110, no 42, 21299-21310 p.Article in journal (Refereed) Published
Abstract [en]

Density functional theory calculations of active site mutants are used to gain insights into the reaction mechanism of the soluble epoxide hydrolases (sEHs). The quantum chemical model is based on the X-ray crystal structure of the human soluble epoxide hydrolase. The role of two conserved active site tyrosines is explored through in silico single and double mutations to phenylalanine. Full potential energy curves for hydrolysis of (1S,2S)-beta-methylstyrene oxide are presented. The results indicate that the two active site tyrosines act in concert to lower the activation barrier for the alkylation step. For the wild-type and three different tyrosine mutant models, the regioselectivity of epoxide opening is compared for the substrates (1S,2S)-beta-methylstyrene oxide and (S)-styrene oxide. An additional part of our study focuses on the importance of the catalytic histidine for the alkylation half-reaction. Different models are presented to explore the protonation state of the catalytic histidine in the alkylation step and to evaluate the possibility of an interaction between the nucleophilic aspartate and the catalytic histidine.

Keyword
Enzyme kinetics; Hydrolysis; Mutagenesis; Potential energy; Probability density function; Quantum theory; Activation barriers; Active site tyrosines; Potential energy curves; Silico singles
National Category
Chemical Sciences
Identifiers
urn:nbn:se:kth:diva-8105 (URN)10.1021/jp063830t (DOI)000241381600086 ()2-s2.0-33751280486 (Scopus ID)
Note
QC 20100811Available from: 2008-03-18 Created: 2008-03-18 Last updated: 2010-08-11Bibliographically approved
4. A theoretical study of th azidolysis and cyanolysis of epozides by haloalcohol dehalogenase
Open this publication in new window or tab >>A theoretical study of th azidolysis and cyanolysis of epozides by haloalcohol dehalogenase
(English)Manuscript (Other academic)
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-7171 (URN)
Note
QC 20101108Available from: 2007-05-22 Created: 2007-05-22 Last updated: 2010-11-08Bibliographically approved

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Citation style
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