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Cyanolysis and Azidolysis of Epoxides by Haloalcohol Dehalogenase: Theoretical Study of the Reaction Mechanism and Origins of Regioselectivity
KTH, School of Biotechnology (BIO), Theoretical Chemistry.
KTH, School of Biotechnology (BIO), Theoretical Chemistry.
2008 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 47, no 17, 4973-4982 p.Article in journal (Refereed) Published
Abstract [en]

Haloalcohol dehalogenase HheC catalyzes the reversible dehalogenation of vicinal haloalcohols to form epoxides and free halides. In addition, HheC is able to catalyze the irreversible and highly regioselective ring-opening of epoxides with nonhalide nucleophiles, such as CN- and N-3(-). For azidolysis of aromatic epoxides, the regioselectivity observed with HheC is opposite to the regioselectivity of the nonenzymatic epoxide-opening. This, together with a relatively broad substrate specificity, makes HheC a promising tool for biocatalytic applications. We have designed large quantum chemical models of the HheC active site and used density functional theory to study the reaction mechanism of the HheC-catalyzed ring-opening of (R)-styrene oxide with the nucleophiles CN- and N3-. Both the cyanolysis and the azidolysis reactions are shown to take place in a single concerted step. The results support the suggested role of the putative Ser132-Tyr145-Arg149 catalytic triad, where Tyr145 acts as a general acid, donating a proton to the substrate, and Arg149 interacts with Tyr145 and facilitates proton abstraction, while Ser132 positions the substrate and reduces the barrier for epoxide opening through interaction with the emerging oxyanion of the substrate. We have also studied the regioselectivity of (R)-styrene oxide opening for both the cyanolysis and the azidolysis reactions. The employed active site model was shown to be able to reproduce the experimentally observed beta-regioselectivity of HheC. In silico mutations of various groups in the HheC active site model were performed to elucidate the important factors governing the regioselectivity.

Place, publisher, year, edition, pages
2008. Vol. 47, no 17, 4973-4982 p.
Keyword [en]
Alcohols; Dehalogenation; Density functional theory; Mathematical models; Quantum chemistry; Reaction kinetics; Regioselectivity
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:kth:diva-8107DOI: 10.1021/bi800001rISI: 000255164700013OAI: oai:DiVA.org:kth-8107DiVA: diva2:13338
Note
QC 20100811. Uppdaterad från in press till published (20100811).Available from: 2008-03-18 Created: 2008-03-18 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Nitrile Hydratases and Epoxide-Transforming Enzymes: Quantum Chemical Modeling of Reaction Mechanisms and Selectivities
Open this publication in new window or tab >>Nitrile Hydratases and Epoxide-Transforming Enzymes: Quantum Chemical Modeling of Reaction Mechanisms and Selectivities
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Quantum chemical studies of enzymatic reactions are able to provide detailed insight into mechanisms and catalytic strategies. The energetic feasibility of proposed mechanisms can be established, and new possible reaction pathways can be put forward. The role of the involved active site residues can be analyzed in detail and the origins for experimentally observed selectivities can be investigated. Density functional theory (DFT), in particular the hybrid functional B3LYP, is the method of choice in this kind of studies.

In this thesis, the reaction mechanisms of several enzymes have been explored using the B3LYP functional. The studied enzymes include limonene epoxide hydrolase (LEH), soluble epoxide hydrolase (sEH), haloalcohol dehalogenase (HheC), and nitrile hydratase (NHase). Transition states and intermediates along various reaction pathways were optimized and evaluated.

For the three epoxide-transforming enzymes, the role of the proposed catalytic residues could be confirmed. Analysis of in silico mutations helped to quantify the effect of various functional groups on the barriers and regioselectivities of epoxide opening. A detailed analysis of the factors governing the enzymatic regioselectivities is given.

For nitrile hydratase, various putative first- and second-shell mechanisms have been studied. Active site models based on both the Co(III)-NHase and the Fe(III)-NHase were employed. The studied mechanisms include general base-catalyzed reaction pathways with water as nucleophile as well as two pathways involving cysteine-sulfenate as nucleophile. Several computed mechanisms exhibit similar barriers, making it difficult to pinpoint the true NHase mechanism.

Place, publisher, year, edition, pages
Stockholm: KTH, 2008. x, 74 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2008:1
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-4668 (URN)978-91-7178-885-6 (ISBN)
Public defence
2008-04-04, FB52, AlbaNova, Roslagstullsbacken 21, Stockholm, 10:30
Opponent
Supervisors
Note
QC 20100811Available from: 2008-03-18 Created: 2008-03-18 Last updated: 2010-08-11Bibliographically approved

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