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Quantum Chemical Study of Dual-Substrate Recognition in ω-Transaminase
Stockholm University, Faculty of Science, Department of Organic Chemistry.
Stockholm University, Faculty of Science, Department of Organic Chemistry.
Stockholm University, Faculty of Science, Department of Organic Chemistry.
2017 (English)In: ACS Omega, E-ISSN 2470-1343, Vol. 2, no 3, 890-898 p.Article in journal (Refereed) Published
Abstract [en]

ω-Transaminases are attractive biocatalysts for the production of chiral amines. These enzymes usually have a broad substrate range. Their substrates include hydrophobic amines as well as amino acids, a feature referred to as dual-substrate recognition. In the present study, the reaction mechanism for the half-transamination of L-alanine to pyruvate in (S)-selective Chromobacterium violaceum ω-transaminase is investigated using density functional theory calculations. The role of a flexible arginine residue, Arg416, in the dual-substrate recognition is investigated by employing two active-site models, one including this residue and one lacking it. The results of this study are compared to those of the mechanism of the conversion of (S)-1-phenylethylamine to acetophenone. The calculations suggest that the deaminations of amino acids and hydrophobic amines follow essentially the same mechanism, but the energetics of the reactions differ significantly. It is shown that the amine is kinetically favored in the half-transamination of L-alanine/pyruvate, whereas the ketone is kinetically favored in the half-transamination of (S)-1-phenylethylamine/acetophenone. The calculations further support the proposal that the arginine residue facilitates the dual-substrate recognition by functioning as an arginine switch, where the side chain is positioned inside or outside of the active site depending on the substrate. Arg416 participates in the binding of L-alanine by forming a salt bridge to the carboxylate moiety, whereas the conversion of (S)-1-phenylethylamine is feasible in the absence of Arg416, which here represents the case in which the side chain of Arg416 is positioned outside of the active site.

Place, publisher, year, edition, pages
2017. Vol. 2, no 3, 890-898 p.
National Category
Organic Chemistry
Research subject
Organic Chemistry
Identifiers
URN: urn:nbn:se:su:diva-141316DOI: 10.1021/acsomega.6b00376ISI: 000399309700015OAI: oai:DiVA.org:su-141316DiVA: diva2:1086613
Funder
Swedish Research CouncilKnut and Alice Wallenberg Foundation
Available from: 2017-04-03 Created: 2017-04-03 Last updated: 2017-05-29Bibliographically approved
In thesis
1. Quantum Chemical Studies of Enzymatic Reaction Mechanisms
Open this publication in new window or tab >>Quantum Chemical Studies of Enzymatic Reaction Mechanisms
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Computer modeling of enzymes is a valuable complement to experiments. Quantum chemical studies of enzymatic reactions can provide a detailed description of the reaction mechanism and elucidate the roles of various residues in the active site. Different reaction pathways can be analyzed, and their feasibility be established based on calculated energy barriers.

In the present thesis, density functional theory has been used to study the active sites and reaction mechanisms of three different enzymes, cytosine deaminase (CDA) from Escherichia coli, ω-transaminase from Chromobacterium violaceum (Cv-ωTA) and dinitrogenase reductase-activating glycohydrolase (DraG) from Rhodospirillum rubrum. The cluster approach has been employed to design models of the active sites based on available crystal structures. The geometries and energies of transition states and intermediates along various reaction pathways have been calculated, and used to construct the energy graphs of the reactions.

In the study of CDA (Paper I), two different tautomers of a histidine residue were considered. The obtained reaction mechanism was found to support the main features of the previously proposed mechanism. The sequence of the events was established, and the residues needed for the proton transfer steps were elucidated.

In the study of Cv-ωTA (Paper II and Paper III), two active site models were employed to study the conversion of two different substrates, a hydrophobic amine and an amino acid. Differences and similarities in the reaction mechanisms of the two substrates were established, and the role of an arginine residue in the dual substrate recognition was confirmed.

In the study of DraG (Paper IV), two different substrate-binding modes and two different protonation states of an aspartate residue were considered. The coordination of the first-shell ligands and the substrate to the two manganese ions in the active site was characterized, and a possible proton donor in the first step of the proposed reaction mechanism was identified.

Place, publisher, year, edition, pages
Stockholm: Department of Organic Chemistry, Stockholm University, 2017. 64 p.
Keyword
density functional theory, B3LYP, enzyme, cluster approach, mechanism, zinc, manganese, cytosine deaminase, ω-transaminase, dinitrogenase reductase-activating glycohydrolase, dual substrate recognition
National Category
Organic Chemistry
Research subject
Organic Chemistry
Identifiers
urn:nbn:se:su:diva-141321 (URN)978-91-7649-764-7 (ISBN)978-91-7649-765-4 (ISBN)
Public defence
2017-05-23, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 14:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.

Available from: 2017-04-27 Created: 2017-04-03 Last updated: 2017-04-27Bibliographically approved

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