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  • 1.
    Amrein, Beat A.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Bauer, Paul
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Duarte, Fernanda
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Janfalk Carlsson, Åsa
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Naworyta, Agata
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Mowbray, Sherry L.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Kamerlin, Shina C. L.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Expanding the catalytic triad in epoxide hydrolases and related enzymes2015In: ACS Catalysis, ISSN 2155-5435, E-ISSN 2155-5435, Vol. 5, no 10, p. 5702-5713Article in journal (Refereed)
    Abstract [en]

    Potato epoxide hydrolase 1 exhibits rich enantio- and regioselectivity in the hydrolysis of a broadrange of substrates. The enzyme can be engineered to increase the yield of optically pureproducts, as a result of changes in both enantio- and regioselectivity. It is thus highly attractive inbiocatalysis, particularly for the generation of enantiopure fine chemicals and pharmaceuticals.The present work aims to establish the principles underlying the activity and selectivity of theenzyme through a combined computational, structural, and kinetic study, using the substratetrans-stilbene oxide as a model system. Extensive empirical valence bond simulations have beenperformed on the wild-type enzyme together with several experimentally characterized mutants.We are able to computationally reproduce the differences in activities between differentstereoisomers of the substrate, and the effects of mutations in several active-site residues. Inaddition, our results indicate the involvement of a previously neglected residue, H104, which iselectrostatically linked to the general base, H300. We find that this residue, which is highlyconserved in epoxide hydrolases and related hydrolytic enzymes, needs to be in its protonatedform in order to provide charge balance in an otherwise negatively-charged active site. Our datashow that unless the active-site charge balance is correctly treated in simulations, it is notpossible to generate a physically meaningful model for the enzyme that can accurately reproduceactivity and selectivity trends. We also expand our understanding of other catalytic residues,demonstrating in particular the role of a non-canonical residue, E35, as a “backup-base” in theabsence of H300. Our results provide a detailed view of the main factors driving catalysis andregioselectivity in this enzyme, and identify targets for subsequent enzyme design efforts.

  • 2.
    Barrozo, Alexandre
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Duarte, Fernanda
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Bauer, Paul
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Carvalho, Alexandra T. P.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Kamerlin, Shina C. Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Cooperative Electrostatic Interactions Drive Functional Evolution in the Alkaline Phosphatase Superfamily2015In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 137, no 28, p. 9061-9076Article in journal (Refereed)
    Abstract [en]

    It is becoming widely accepted that catalytic promiscuity, i.e., the ability of a single enzyme to catalyze the turnover of multiple, chemically distinct substrates, plays a key role in the evolution of new enzyme functions. In this context, the members of the alkaline phosphatase superfamily have been extensively studied as model systems in order to understand the phenomenon of enzyme multifunctionality. In the present work, we model the selectivity of two multiply promiscuous members of this superfamily, namely the phosphonate monoester hydrolases from Burkholderia caryophylli and Rhizobium leguminosarum. We have performed extensive simulations of the enzymatic reaction of both wild-type enzymes and several experimentally characterized mutants. Our computational models are in agreement with key experimental observables, such as the observed activities of the wild-type enzymes, qualitative interpretations of experimental pH-rate profiles, and activity trends among several active site mutants. In all cases the substrates of interest bind to the enzyme in similar conformations, with largely unperturbed transition states from their corresponding analogues in aqueous solution. Examination of transition-state geometries and the contribution of individual residues to the calculated activation barriers suggest that the broad promiscuity of these enzymes arises from cooperative electrostatic interactions in the active site, allowing each enzyme to adapt to the electrostatic needs of different substrates. By comparing the structural and electrostatic features of several alkaline phosphatases, we suggest that this phenomenon is a generalized feature driving selectivity and promiscuity within this superfamily and can be in turn used for artificial enzyme design.

  • 3.
    Duarte, Fernanda
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Amrein, Beat A.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Blaha-Nelson, David
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Kamerlin, Shina Caroline Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Recent advances in QM/MM free energy calculations using reference potentials2015In: Biochimica et Biophysica Acta - General Subjects, ISSN 0304-4165, E-ISSN 1872-8006, Vol. 1850, no 5, p. 954-965Article, review/survey (Refereed)
    Abstract [en]

    Background: Recent years have seen enormous progress in the development of methods for modeling (bio)molecular systems. This has allowed for the simulation of ever larger and more complex systems. However, as such complexity increases, the requirements needed for these models to be accurate and physically meaningful become more and more difficult to fulfill. The use of simplified models to describe complex biological systems has long been shown to be an effective way to overcome some of the limitations associated with this computational cost in a rational way. Scope of review: Hybrid QM/MM approaches have rapidly become one of the most popular computational tools for studying chemical reactivity in biomolecular systems. However, the high cost involved in performing high-level QM calculations has limited the applicability of these approaches when calculating free energies of chemical processes. In this review, we present some of the advances in using reference potentials and mean field approximations to accelerate high-level QM/MM calculations. We present illustrative applications of these approaches and discuss challenges and future perspectives for the field. Major conclusions: The use of physically-based simplifications has shown to effectively reduce the cost of high-level QM/MM calculations. In particular, lower-level reference potentials enable one to reduce the cost of expensive free energy calculations, thus expanding the scope of problems that can be addressed. General significance: As was already demonstrated 40 years ago, the usage of simplified models still allows one to obtain cutting edge results with substantially reduced computational cost. This article is part of a Special Issue entitled Recent developments of molecular dynamics.

  • 4.
    Duarte, Fernanda
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Amrein, Beat Anton
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Kamerlin, Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Modeling catalytic promiscuity in the alkaline phosphatase superfamily2013In: Physical Chemistry, Chemical Physics - PCCP, ISSN 1463-9076, E-ISSN 1463-9084, Vol. 15, no 27, p. 11160-11177Article in journal (Refereed)
    Abstract [en]

    In recent years, it has become increasingly clear that promiscuity plays a key role in the evolution of new enzyme function. This finding has helped to elucidate fundamental aspects of molecular evolution. While there has been extensive experimental work on enzyme promiscuity, computational modeling of the chemical details of such promiscuity has traditionally fallen behind the advances in experimental studies, not least due to the nearly prohibitive computational cost involved in examining multiple substrates with multiple potential mechanisms and binding modes in atomic detail with a reasonable degree of accuracy. However, recent advances in both computational methodologies and power have allowed us to reach a stage in the field where we can start to overcome this problem, and molecular simulations can now provide accurate and efficient descriptions of complex biological systems with substantially less computational cost. This has led to significant advances in our understanding of enzyme function and evolution in a broader sense. Here, we will discuss currently available computational approaches that can allow us to probe the underlying molecular basis for enzyme specificity and selectivity, discussing the inherent strengths and weaknesses of each approach. As a case study, we will discuss recent computational work on different members of the alkaline phosphatase superfamily (AP) using a range of different approaches, showing the complementary insights they have provided. We have selected this particular superfamily, as it poses a number of significant challenges for theory, ranging from the complexity of the actual reaction mechanisms involved to the reliable modeling of the catalytic metal centers, as well as the very large system sizes. We will demonstrate that, through current advances in methodologies, computational tools can provide significant insight into the molecular basis for catalytic promiscuity, and, therefore, in turn, the mechanisms of protein functional evolution.

  • 5.
    Duarte, Fernanda
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Bauer, Paul
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Barrozo, Alexandre
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Amrein, Beat Anton
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Purg, Miha
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Kamerlin, Shina Caroline Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Force Field Independent Metal Parameters Using a Nonbonded Dummy Model2014In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 118, no 16, p. 4351-4362Article in journal (Refereed)
    Abstract [en]

    The cationic dummy atom approach provides a powerful nonbonded description for a range of alkaline-earth and transition-metal centers, capturing both structural and electrostatic effects. In this work we refine existing literature parameters for octahedrally coordinated Mn2+, Zn2+, Mg2+, and Ca2+, as well as providing new parameters for Ni2+, Co2+, and Fe2+. In all the cases, we are able to reproduce both M2+-O distances and experimental solvation free energies, which has not been achieved to date for transition metals using any other model. The parameters have also been tested using two different water models and show consistent performance. Therefore, our parameters are easily transferable to any force field that describes nonbonded interactions using Coulomb and Lennard-Jones potentials. Finally, we demonstrate the stability of our parameters in both the human and Escherichia coli variants of the enzyme glyoxalase 1 as showcase systems, as both enzymes are active with a range of transition metals. The parameters presented in this work provide a valuable resource for the molecular simulation community, as they extend the range of metal ions that can be studied using classical approaches, while also providing a starting point for subsequent parametrization of new metal centers.

  • 6.
    Duarte, Fernanda
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Geng, Ting
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Marloie, Gaël
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Al Hussain, Adel O.
    Williams, Nicholas H.
    Kamerlin, Shina Caroline Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    The Alkaline Hydrolysis of Sulfonate Esters: Challenges in Interpreting Experimental and Theoretical Data2014In: Journal of Organic Chemistry, ISSN 0022-3263, E-ISSN 1520-6904, Vol. 79, no 7, p. 2816-2828Article in journal (Refereed)
    Abstract [en]

    Sulfonate ester hydrolysis has been the subject of recent debate, with experimental evidence interpreted in terms of both stepwise and concerted mechanisms. In particular, a recent study of the alkaline hydrolysis of a series of benzene arylsulfonates (Babtie et al., Org. Biomol. Chem. 10, 2012, 8095) presented a nonlinear Bronsted plot, which was explained in terms of a change from a stepwise mechanism involving a pentavalent intermediate for poorer leaving groups to a fully concerted mechanism for good leaving groups and supported by a theoretical study. In the present work, we have performed a detailed computational study of the hydrolysis of these compounds and find no computational evidence for a thermodynamically stable intermediate for any of these compounds. Additionally, we have extended the experimental data to include pyridine-3-yl benzene sulfonate and its N-oxide and N-methylpyridinium derivatives. Inclusion of these compounds converts the Bronsted plot to a moderately scattered but linear correlation and gives a very good Hammett correlation. These data suggest a concerted pathway for this reaction that proceeds via an early transition state with little bond cleavage to the leaving group, highlighting the care that needs to be taken with the interpretation of experimental and especially theoretical data.

  • 7.
    Duarte, Fernanda
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Gronert, Scott
    Kamerlin, Shina Caroline Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Concerted or Stepwise: How Much Do Free-Energy Landscapes Tell Us about the Mechanisms of Elimination Reactions?2014In: Journal of Organic Chemistry, ISSN 0022-3263, E-ISSN 1520-6904, Vol. 79, no 3, p. 1280-1288Article in journal (Refereed)
    Abstract [en]

    The base-catalyzed dehydration of benzene cis-1,2-dihydrodiols is driven by formation of an aromatic product as well as intermediates potentially stabilized by hyperaromaticity. Experiments exhibit surprising shifts in isotope effects, indicating an unusual mechanistic balance on the E2-E1cB continuum. In this study, both land 2-dimensional free energy surfaces are generated for these compounds with various substituents, using density functional theory and a mixed implicit/explicit solvation model. The computational data help unravel hidden intermediates along the reaction coordinate and provide a novel conceptual framework for distinguishing between competing pathways in this and any other system with borderline reaction mechanisms.

  • 8.
    Duarte, Fernanda
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Kamerlin, Shina C. Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Rationalizing the Catalytic Activity of an Unusual Oxidoreductase (FucO)2013In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 104, no 2, p. 495A-495AArticle in journal (Other academic)
  • 9.
    Duarte, Fernanda
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Williams, Nicholas H
    Kamerlin, Shina C Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Resolving apparent conflicts between theoretical and experimental models of phosphate monoester hydrolysis.2015In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 137, no 3, p. 1081-93Article in journal (Refereed)
    Abstract [en]

    Understanding phosphoryl and sulfuryl transfer is central to many biochemical processes. However, despite decades of experimental and computational studies, a consensus concerning the precise mechanistic details of these reactions has yet to be reached. In this work we perform a detailed comparative theoretical study of the hydrolysis of p-nitrophenyl phosphate, methyl phosphate and p-nitrophenyl sulfate, all of which have served as key model systems for understanding phosphoryl and sulfuryl transfer reactions, respectively. We demonstrate the existence of energetically similar but mechanistically distinct possibilities for phosphate monoester hydrolysis. The calculated kinetic isotope effects for p-nitrophenyl phosphate provide a means to discriminate between substrate- and solvent-assisted pathways of phosphate monoester hydrolysis, and show that the solvent-assisted pathway dominates in solution. This preferred mechanism for p-nitrophenyl phosphate hydrolysis is difficult to find computationally due to the limitations of compressing multiple bonding changes onto a 2-dimensional energy surface. This problem is compounded by the need to include implicit solvation to at least microsolvate the system and stabilize the highly charged species. In contrast, methyl phosphate hydrolysis shows a preference for a substrate-assisted mechanism. For p-nitrophenyl sulfate hydrolysis there is only one viable reaction pathway, which is similar to the solvent-assisted pathway for phosphate hydrolysis, and the substrate-assisted pathway is not accessible. Overall, our results provide a unifying mechanistic framework that is consistent with the experimentally measured kinetic isotope effects and reconciles the discrepancies between theoretical and experimental models for these biochemically ubiquitous classes of reaction.

  • 10.
    Miton, Charlotte M.
    et al.
    Univ Cambridge, Dept Biochem, Cambridge CB2 1GA, England;Univ British Columbia, Michael Smith Labs, Vancouver, BC V6T 1Z4, Canada.
    Jonas, Stefanie
    Univ Cambridge, Dept Biochem, Cambridge CB2 1GA, England;Swiss Fed Inst Technol, Dept Biol, Inst Biochem, CH-8093 Zurich, Switzerland.
    Fischer, Gerhard
    Univ Cambridge, Dept Biochem, Cambridge CB2 1GA, England.
    Duarte, Fernanda
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC. Univ Edinburgh, EaStCHEM Sch Chem, Edinburgh EH9 3FJ, Midlothian, Scotland.
    Mohamed, Mark F.
    Univ Cambridge, Dept Biochem, Cambridge CB2 1GA, England.
    van Loo, Bert
    Univ Cambridge, Dept Biochem, Cambridge CB2 1GA, England;Westfal Wilhelms Univ, Inst Evolut & Biodivers, D-48149 Munster, Germany.
    Kintses, Balint
    Univ Cambridge, Dept Biochem, Cambridge CB2 1GA, England;Hungarian Acad Sci, Biol Res Ctr, Inst Biochem, Synthet & Syst Biol Unit, H-6726 Szeged, Hungary.
    Kamerlin, Shina C. Lynn
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Tokuriki, Nobuhiko
    Univ Cambridge, Dept Biochem, Cambridge CB2 1GA, England;Univ British Columbia, Michael Smith Labs, Vancouver, BC V6T 1Z4, Canada.
    Hyvonen, Marko
    Univ Cambridge, Dept Biochem, Cambridge CB2 1GA, England.
    Hollfelder, Florian
    Univ Cambridge, Dept Biochem, Cambridge CB2 1GA, England.
    Evolutionary repurposing of a sulfatase: A new Michaelis complex leads to efficient transition state charge offset2018In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 115, no 31, p. E7293-E7302Article in journal (Refereed)
    Abstract [en]

    The recruitment and evolutionary optimization of promiscuous enzymes is key to the rapid adaptation of organisms to changing environments. Our understanding of the precise mechanisms underlying enzyme repurposing is, however, limited: What are the active-site features that enable the molecular recognition of multiple substrates with contrasting catalytic requirements? To gain insights into the molecular determinants of adaptation in promiscuous enzymes, we performed the laboratory evolution of an arylsulfatase to improve its initially weak phenylphosphonate hydrolase activity. The evolutionary trajectory led to a 100,000-fold enhancement of phenylphosphonate hydrolysis, while the native sulfate and promiscuous phosphate mono-and diester hydrolyses were only marginally affected (<= 50-fold). Structural, kinetic, and in silico characterizations of the evolutionary intermediates revealed that two key mutations, T50A and M72V, locally reshaped the active site, improving access to the catalytic machinery for the phosphonate. Measured transition state (TS) charge changes along the trajectory suggest the creation of a new Michaelis complex (E.S, enzyme-substrate), with enhanced leaving group stabilization in the TS for the promiscuous phosphonate (beta(leaving) (group) from -1.08 to -0.42). Rather than altering the catalytic machinery, evolutionary repurposing was achieved by fine-tuning the molecular recognition of the phosphonate in the Michaelis complex, and by extension, also in the TS. This molecular scenario constitutes a mechanistic alternative to adaptation solely based on enzyme flexibility and conformational selection. Instead, rapid functional transitions between distinct chemical reactions rely on the high reactivity of permissive active-site architectures that allow multiple substrate binding modes.

  • 11.
    Repic, Matej
    et al.
    Laboratory for Biocomputing and Bioinformatics, National Institute of Chemistry, Ljubljana, Slovenia.
    Vianello, Robert
    Quantum Organic Chemistry Group, Ruđer Bošković Institute, Zagreb, Croatia.
    Purg, Miha
    Laboratory for Biocomputing and Bioinformatics, National Institute of Chemistry, Ljubljana, Slovenia.
    Duarte, Fernanda
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Bauer, Paul
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Kamerlin, Lynn Shina Caroline
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Mavri, Janez
    Laboratory for Biocomputing and Bioinformatics, National Institute of Chemistry, Ljubljana, Slovenia.
    Empirical valence bond simulations of the hydride transfer step in the monoamine oxidase B catalyzed metabolism of dopamine2014In: Proteins: Structure, Function, and Bioinformatics, ISSN 0887-3585, E-ISSN 1097-0134, Vol. 82, no 12, p. 3347-3355Article in journal (Refereed)
    Abstract [en]

    Monoamine oxidases (MAOs) A and B are flavoenzymes responsible for the metabolism of biogenic amines such as dopamine, serotonin and noradrenaline. In this work, we present a comprehensive study of the rate-limiting step of dopamine degradation by MAO B, which consists in the hydride transfer from the methylene group of the substrate to the flavin moiety of the FAD prosthetic group. This article builds on our previous quantum chemical study of the same reaction using a cluster model (Vianello et al., Eur J Org Chem 2012; 7057), but now considering the full dimensionality of the hydrated enzyme with extensive configurational sampling. We show that MAO B is specifically tuned to catalyze the hydride transfer step from the substrate to the flavin moiety of the FAD prosthetic group and that it lowers the activation barrier by 12.3 kcal mol(-1) compared to the same reaction in aqueous solution, a rate enhancement of more than nine orders of magnitude. Taking into account the deprotonation of the substrate prior to the hydride transfer reaction, the activation barrier in the enzyme is calculated to be 16.1 kcal mol(-1), in excellent agreement with the experimental value of 16.5 kcal mol(-1). Additionally, we demonstrate that the protonation state of the active site residue Lys296 does not have an influence on the hydride transfer reaction.

  • 12. Tirel, Emmanuel Y.
    et al.
    Bellamy, Zoe
    Adams, Harry
    Lebrun, Vincent
    Duarte, Fernanda
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Williams, Nicholas H.
    Catalytic Zinc Complexes for Phosphate Diester Hydrolysis2014In: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 53, no 31, p. 8246-8250Article in journal (Refereed)
    Abstract [en]

    Creating efficient artificial catalysts that can compete with biocatalysis has been an enduring challenge which has yet to be met. Reported herein is the synthesis and characterization of a series of zinc complexes designed to catalyze the hydrolysis of phosphate diesters. By introducing a hydrated aldehyde into the ligand we achieve turnover for DNA-like substrates which, combined with ligand methylation, increases reactivity by two orders of magnitude. In contrast to current orthodoxy and mechanistic explanations, we propose a mechanism where the nucleophile is not coordinated to the metal ion, but involves a tautomer with a more effective Lewis acid and more reactive nucleophile. This data suggests a new strategy for creating more efficient metal ion based catalysts, and highlights a possible mode of action for metalloenzymes.

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