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A quantum chemical study of the ω-transaminase reaction mechanism
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.
2015 (English)In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 13, no 31, 8453-8464 p.Article in journal (Refereed) Published
Abstract [en]

ω-Transaminases are valuable tools in biocatalysis due to their stereospecificity and their broad substrate range. In the present study, the reaction mechanism of Chromobacterium violaceum ω-transaminase is investigated by means of density functional theory calculations. A large active site model is designed based on the recent X-ray crystal structure. The detailed energy profile for the half-transamination of (S)-1-phenylethylamine to acetophenone is calculated and the involved transition states and intermediates are characterized. The model suggests that the amino substrate forms an external aldimine with the coenzyme pyridoxal-5′-phosphate (PLP), through geminal diamine intermediates. The external aldimine is then deprotonated in the rate-determining step, forming a planar quinonoid intermediate. A ketimine is then formed, after which a hemiaminal is produced by the addition of water. Subsequently, the ketone product is obtained together with pyridoxamine-5′-phosphate (PMP). In the studied half-transamination reaction the ketone product is kinetically favored. The mechanism presented here will be valuable to enhance rational and semi-rational design of engineered enzyme variants in the development of ω-transaminase chemistry.

Place, publisher, year, edition, pages
2015. Vol. 13, no 31, 8453-8464 p.
National Category
Organic Chemistry
Research subject
Organic Chemistry
Identifiers
URN: urn:nbn:se:su:diva-120490DOI: 10.1039/c5ob00690bISI: 000358733100011OAI: oai:DiVA.org:su-120490DiVA: diva2:852851
Funder
Swedish Research CouncilKnut and Alice Wallenberg Foundation
Available from: 2015-09-10 Created: 2015-09-10 Last updated: 2017-12-04Bibliographically 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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