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Quantum Chemical Studies of Enzymatic Reaction Mechanisms
Stockholm University, Faculty of Science, Department of Organic Chemistry.
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 [en]
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: urn:nbn:se:su:diva-141321ISBN: 978-91-7649-764-7 (print)ISBN: 978-91-7649-765-4 (electronic)OAI: oai:DiVA.org:su-141321DiVA: diva2:1086730
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
List of papers
1. Reaction Mechanism of Zinc-Dependent Cytosine Deaminase from Escherichia coli: A Quantum-Chemical Study
Open this publication in new window or tab >>Reaction Mechanism of Zinc-Dependent Cytosine Deaminase from Escherichia coli: A Quantum-Chemical Study
2014 (English)In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 118, no 21, 5644-5652 p.Article in journal (Refereed) Published
Abstract [en]

The reaction mechanism of cytosine deaminase from Escherichia coli is studied using density functional theory. This zinc-dependent enzyme catalyzes the deamination of cytosine to form uracil and ammonia. The calculations give a detailed description of the catalytic mechanism and establish the role of important active-site residues. It is shown that Glu217 is essential for the initial deprotonation of the metal-bound water nucleophile and the subsequent protonation of the substrate. It is also demonstrated that His246 is unlikely to function as a proton shuttle in the nucleophile activation step, as previously proposed. The steps that follow are nucleophilic attack by the metal-bound hydroxide, protonation of the leaving group assisted by Asp313, and C-N bond cleavage. The calculated overall barrier is in good agreement with the experimental findings. Finally, the calculations reproduce the experimentally determined inverse solvent deuterium isotope effect, which further corroborates the suggested reaction mechanism.

National Category
Organic Chemistry
Research subject
Organic Chemistry
Identifiers
urn:nbn:se:su:diva-105913 (URN)10.1021/jp501228s (DOI)000336771100003 ()
Funder
Swedish Research CouncilKnut and Alice Wallenberg Foundation
Note

AuthorCount:3;

Available from: 2014-07-08 Created: 2014-07-08 Last updated: 2017-04-03Bibliographically approved
2. A quantum chemical study of the ω-transaminase reaction mechanism
Open this publication in new window or tab >>A quantum chemical study of the ω-transaminase reaction mechanism
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.

National Category
Organic Chemistry
Research subject
Organic Chemistry
Identifiers
urn:nbn:se:su:diva-120490 (URN)10.1039/c5ob00690b (DOI)000358733100011 ()
Funder
Swedish Research CouncilKnut and Alice Wallenberg Foundation
Available from: 2015-09-10 Created: 2015-09-10 Last updated: 2017-04-03Bibliographically approved
3. Quantum Chemical Study of Dual-Substrate Recognition in ω-Transaminase
Open this publication in new window or tab >>Quantum Chemical Study of Dual-Substrate Recognition in ω-Transaminase
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.

National Category
Organic Chemistry
Research subject
Organic Chemistry
Identifiers
urn:nbn:se:su:diva-141316 (URN)10.1021/acsomega.6b00376 (DOI)000399309700015 ()
Funder
Swedish Research CouncilKnut and Alice Wallenberg Foundation
Available from: 2017-04-03 Created: 2017-04-03 Last updated: 2017-05-29Bibliographically approved
4. Insights from Quantum Chemical Calculations into Active Site Structure and Reaction Mechanism of Manganese-Dependent Dinitrogenase Reductase-Activating Glycohydrolase
Open this publication in new window or tab >>Insights from Quantum Chemical Calculations into Active Site Structure and Reaction Mechanism of Manganese-Dependent Dinitrogenase Reductase-Activating Glycohydrolase
(English)Manuscript (preprint) (Other academic)
National Category
Organic Chemistry
Research subject
Organic Chemistry
Identifiers
urn:nbn:se:su:diva-141319 (URN)
Available from: 2017-04-03 Created: 2017-04-03 Last updated: 2017-04-03Bibliographically approved

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