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Bauer, Paul
Publications (10 of 15) Show all publications
Mydy, L. S., Cristobal, J. R., Katigbak, R. D., Bauer, P., Reyes, A. C., Kamerlin, S. C., . . . Gulick, A. M. (2019). Human Glycerol 3-Phosphate Dehydrogenase: X-ray Crystal Structures That Guide the Interpretation of Mutagenesis Studies. Biochemistry, 58(8), 1061-1073
Open this publication in new window or tab >>Human Glycerol 3-Phosphate Dehydrogenase: X-ray Crystal Structures That Guide the Interpretation of Mutagenesis Studies
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2019 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 58, no 8, p. 1061-1073Article in journal (Refereed) Published
Abstract [en]

Human liver glycerol 3-phosphate dehydrogenase (hlGPDH) catalyzes the reduction of dihydroxyacetone phosphate (DHAP) to form glycerol 3-phosphate, using the binding energy associated with the nonreacting phosphodianion of the substrate to properly orient the enzyme-substrate complex within the active site. Herein, we report the crystal structures for unliganded, binary E.NAD, and ternary E.NAD.DHAP complexes of wild type hlGPDH, illustrating a new position of DHAP, and probe the kinetics of multiple mutant enzymes with natural and truncated substrates. Mutation of Lys120, which is positioned to donate a proton to the carbonyl of DHAP, results in similar increases in the activation barrier to hlGPDH-catlyzed reduction of DHAP and to phosphite dianion-activated reduction of glycolaldehyde, illustrating that these transition states show similar interactions with the cationic K120 side chain. The K120A mutation results in a 5.3 kcal/mol transition state destabilization, and 3.0 kcal/mol of the lost transition state stabilization is rescued by 1.0 M ethylammonium cation. The 6.5 kcal/mol increase in the activation barrier observed for the D260G mutant hlGPDH-catalyzed reaction represents a 3.5 kcal/mol weakening of transition state stabilization by the K120A side chain and a 3.0 kcal/mol weakening of the interactions with other residues. The interactions, at the enzyme active site, between the K120 side chain and the Q295 and R269 side chains were likewise examined by double-mutant analyses. These results provide strong evidence that the enzyme rate acceleration is due mainly or exclusively to transition state stabilization by electrostatic interactions with polar amino acid side chains.

Place, publisher, year, edition, pages
AMER CHEMICAL SOC, 2019
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-379583 (URN)10.1021/acs.biochem.8b01103 (DOI)000460199700009 ()30640445 (PubMedID)
Funder
Swedish Research Council, 2015-04928
Available from: 2019-03-25 Created: 2019-03-25 Last updated: 2019-03-25Bibliographically approved
Marsavelski, A., Petrovic, D., Bauer, P., Vianello, R. & Kamerlin, S. C. (2018). Empirical Valence Bond Simulations Suggest a Direct Hydride Transfer Mechanism for Human Diamine Oxidase. ACS Omega, 3(4), 3665-3674
Open this publication in new window or tab >>Empirical Valence Bond Simulations Suggest a Direct Hydride Transfer Mechanism for Human Diamine Oxidase
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2018 (English)In: ACS Omega, ISSN 2470-1343, Vol. 3, no 4, p. 3665-3674Article in journal (Refereed) Published
Abstract [en]

Diamine oxidase (DAO) is an enzyme involved in the regulation of cell proliferation and the immune response. This enzyme performs oxidative deamination in the catabolism of biogenic amines, including, among others, histamine, putrescine, spermidine, and spermine. The mechanistic details underlying the reductive half-reaction of the DAO-catalyzed oxidative deamination which leads to the reduced enzyme cofactor and the aldehyde product are, however, still under debate. The catalytic mechanism was proposed to involve a prototropic shift from the substrateSchiff base to the product-Schiff base, which includes the ratelimiting cleavage of the C alpha-H bond by the conserved catalytic aspartate. Our detailed mechanistic study, performed using a combined quantum chemical cluster approach with empirical valence bond simulations, suggests that the rate-limiting cleavage of the C alpha-H bond involves direct hydride transfer to the topaquinone cofactor. a mechanism that does not involve the formation of a Schiff base. Additional investigation of the D373E and D373N variants supported the hypothesis that the conserved catalytic aspartate is indeed essential for the reaction; however, it does not appear to serve as the catalytic base, as previously suggested. Rather, the electrostatic contributions of the most significant residues (including D373), together with the proximity of the Cu2+ cation to the reaction site, lower the activation barrier to drive the chemical reaction.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-353209 (URN)10.1021/acsomega.8b00346 (DOI)000430200300005 ()
Funder
Swedish Research Council, 2015-04928
Available from: 2018-06-13 Created: 2018-06-13 Last updated: 2018-12-03Bibliographically approved
Janfalk Carlsson, Å., Bauer, P., Dobritzsch, D., Kamerlin, S. C. & Widersten, M. (2018). Epoxide Hydrolysis as a Model System for Understanding Flux Through a Branched Reaction Scheme. IUCrJ, 5(3), 269-282
Open this publication in new window or tab >>Epoxide Hydrolysis as a Model System for Understanding Flux Through a Branched Reaction Scheme
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2018 (English)In: IUCrJ, ISSN 0972-6918, E-ISSN 2052-2525, Vol. 5, no 3, p. 269-282Article in journal (Refereed) Published
Abstract [en]

The epoxide hydrolase StEH1 catalyzes the hydrolysis of trans-methylstyrene oxide to 1-phenyl­propane-1,2-diol. The (S,S)-epoxide is exclusively transformed into the (1R,2S)-diol, while hydrolysis of the (R,R)-epoxide results in a mixture of product enantiomers. In order to understand the differences in the stereoconfigurations of the products, the reactions were studied kinetically during both the pre-steady-state and steady-state phases. A number of closely related StEH1 variants were analyzed in parallel, and the results were rationalized by structure–activity analysis using the available crystal structures of all tested enzyme variants. Finally, empirical valence-bond simulations were performed in order to provide additional insight into the observed kinetic behaviour and ratios of the diol product enantiomers. These combined data allow us to present a model for the flux through the catalyzed reactions. With the (R,R)-epoxide, ring opening may occur at either C atom and with similar energy barriers for hydrolysis, resulting in a mixture of diol enantiomer products. However, with the (S,S)-epoxide, although either epoxide C atom may react to form the covalent enzyme intermediate, only the pro-(R,S) alkylenzyme is amenable to subsequent hydrolysis. Previously contradictory observations from kinetics experiments as well as product ratios can therefore now be explained for this biocatalytically relevant enzyme.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-343750 (URN)10.1107/S2052252518003573 (DOI)000431151300004 ()29755743 (PubMedID)
Funder
Swedish Research CouncilEU, FP7, Seventh Framework Programme
Available from: 2018-03-01 Created: 2018-03-01 Last updated: 2018-12-03Bibliographically approved
Bauer, P., Barrozo, A., Purg, M., Amrein, B. A., Esguerra, M., Wilson, P. B., . . . Kamerlin, S. C. L. (2018). Q6: A comprehensive toolkit for empirical valence bond and related free energy calculations. SoftwareX, 388-395
Open this publication in new window or tab >>Q6: A comprehensive toolkit for empirical valence bond and related free energy calculations
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2018 (English)In: SoftwareX, ISSN 2352-7110, p. 388-395Article in journal (Refereed) Published
Abstract [en]

Atomistic simulations have become one of the main approaches to study the chemistry and dynamicsof biomolecular systems in solution. Chemical modelling is a powerful way to understand biochemistry,with a number of different programs available to perform specialized calculations. We present here Q6, anew version of the Q software package, which is a generalized package for empirical valence bond, linearinteraction energy, and other free energy calculations. In addition to general technical improvements, Q6extends the reach of the EVB implementation to fast approximations of quantum effects, extended solventdescriptions and quick estimation of the contributions of individual residues to changes in the activationfree energy of reactions.

National Category
Software Engineering
Identifiers
urn:nbn:se:uu:diva-360517 (URN)10.1016/j.softx.2017.12.001 (DOI)000457139300064 ()
Funder
Swedish Research Council, 2014-3688Swedish Research Council, 2014-2118Swedish Research Council, 2015-04928
Available from: 2018-09-14 Created: 2018-09-14 Last updated: 2019-02-25Bibliographically approved
Maurer, D., Enugala, T. R., Hamnevik, E., Bauer, P., Lüking, M., Petrovic, D., . . . Widersten, M. (2018). Stereo- and Regioselectivity in Catalyzed Transformation of a 1,2-Disubstituted Vicinal Diol and the Corresponding Diketone by Wild Type and Laboratory Evolved Alcohol Dehydrogenases. ACS Catalysis, 8(8), 7526-7538
Open this publication in new window or tab >>Stereo- and Regioselectivity in Catalyzed Transformation of a 1,2-Disubstituted Vicinal Diol and the Corresponding Diketone by Wild Type and Laboratory Evolved Alcohol Dehydrogenases
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2018 (English)In: ACS Catalysis, ISSN 2155-5435, E-ISSN 2155-5435, Vol. 8, no 8, p. 7526-7538Article in journal (Refereed) Published
Abstract [en]

ADH-A from Rhodococcus ruber DSM 44541 catalyzes the oxidation of (S)-1-phenylethanol 3000-fold more efficiently as compared with the 2-hydroxylated derivative (R)-phenylethane-1,2-diol. The enzyme is also highly selective for sec-alcohols with comparably low activities with the corresponding primary alcohols. When challenged with a substrate containing two secondary alcohols, such as 1-phenylpropane-(1R,2S)-diol, ADH-A favors the oxidation of the benzylic carbon of this alcohol. The catalytic efficiency, however, is modest in comparison to the activity with (S)-1-phenylethanol. To investigate the structural requirements for improved oxidation of vicinal diols, we conducted iterative saturation mutagenesis combined with activity screening. A first-generation variant, B1 (Y54G, L119Y) displays a 2-fold higher kcat value with 1-phenylpropane-(1R,2S)-diol and a shift in the cooperative behavior in alcohol binding, from negative in the wild type, to positive in B1, suggesting a shift from a less active enzyme form (T) in the wild type to a more active form (R) in the B1 variant. Also, the regiopreference changed to favor oxidation of C-2. A second-generation variant, B1F4 (F43T, Y54G, L119Y, F282W), shows further improvement in the turnover and regioselectivity in oxidation of 1-phenylpropane-(1R,2S)-diol. The crystal structures of the B1 and B1F4 variants describe the structural alterations to the active site, the most significant of which is a repositioning of a Tyr side-chain located distal to the coenzyme and the catalytic zinc ion. The links between the changes in structures and stereoselectivities are rationalized by molecular dynamics simulations of substrate binding at the respective active sites.

Keywords: alcohol dehydrogenase; alcohol oxidation; biocatalysis; crystal structure; directed evolution; enzyme engineering; molecular dynamics simulations; stereoselectivity

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-355854 (URN)10.1021/acscatal.8b01762 (DOI)000441112400074 ()
Funder
Stiftelsen Olle Engkvist ByggmästareSwedish Research Council, 2015-04928Knut and Alice Wallenberg Foundation, KAW 2013.0124EU, FP7, Seventh Framework Programme, 283570Swedish National Infrastructure for Computing (SNIC), 2015/16-12Swedish National Infrastructure for Computing (SNIC), 2016/34-27
Available from: 2018-07-05 Created: 2018-07-05 Last updated: 2018-11-29Bibliographically approved
Hong, N.-S., Petrovic, D., Lee, R., Gryn'ova, G., Purg, M., Saunders, J., . . . Jackson, C. J. (2018). The evolution of multiple active site configurations in a designed enzyme. Nature Communications, 9, Article ID 3900.
Open this publication in new window or tab >>The evolution of multiple active site configurations in a designed enzyme
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2018 (English)In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 9, article id 3900Article in journal (Refereed) Published
Abstract [en]

Developments in computational chemistry, bioinformatics, and laboratory evolution have facilitated the de novo design and catalytic optimization of enzymes. Besides creating useful catalysts, the generation and iterative improvement of designed enzymes can provide valuable insight into the interplay between the many phenomena that have been suggested to contribute to catalysis. In this work, we follow changes in conformational sampling, electrostatic preorganization, and quantum tunneling along the evolutionary trajectory of a designed Kemp eliminase. We observe that in the Kemp Eliminase KE07, instability of the designed active site leads to the emergence of two additional active site configurations. Evolutionary conformational selection then gradually stabilizes the most efficient configuration, leading to an improved enzyme. This work exemplifies the link between conformational plasticity and evolvability and demonstrates that residues remote from the active sites of enzymes play crucial roles in controlling and shaping the active site for efficient catalysis.

Place, publisher, year, edition, pages
Nature Publishing Group, 2018
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-363422 (URN)10.1038/s41467-018-06305-y (DOI)000445560800008 ()30254369 (PubMedID)
Funder
Australian Research Council, DP130102144EU, FP7, Seventh Framework Programme, 306474Knut and Alice Wallenberg Foundation
Available from: 2018-10-18 Created: 2018-10-18 Last updated: 2018-10-18Bibliographically approved
Bauer, P. (2017). Computational modelling of enzyme selectivity. (Doctoral dissertation). Uppsala: Acta Universitatis Upsaliensis
Open this publication in new window or tab >>Computational modelling of enzyme selectivity
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Enantioselective reactions are one of the ways to produce pure chiral compounds. Understanding the basis of this selectivity makes it possible to guide enzyme design towards more efficient catalysts. One approach to study enzymes involved in chiral chemistry is through the use of computational models that are able to simulate the chemical reaction taking place. The potato epoxide hydrolase is one enzyme that is known to be both highly enantioselective, while still being robust upon mutation of residues to change substrate scope. The enzyme was used to investigate the epoxide hydrolysis mechanism for a number of different substrates, using the EVB approach to the reaction both in solution and in several enzyme variants. In addition to this, work has been performed on new ways of performing simulations of divalent transition metals, as well as development of new simulation software.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2017. p. 104
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1530
Keywords
enantiomer, epoxide hydrolase, chiral catalysis, empirical valence bond approach, method development
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-326108 (URN)978-91-513-0005-4 (ISBN)
Public defence
2017-09-13, A1:111 BMC, Husargatan 3, Uppsala, 09:00 (English)
Opponent
Supervisors
Available from: 2017-08-21 Created: 2017-07-02 Last updated: 2018-12-03
Bauer, P., Janfalk Carlsson, Å., Amrein, B. A., Dobritzsch, D., Widersten, M. & Kamerlin, S. C. (2016). Conformational Diversity and Enantioconvergence in Potato Epoxide Hydrolase 1. Organic and biomolecular chemistry, 14(24), 5639-5651
Open this publication in new window or tab >>Conformational Diversity and Enantioconvergence in Potato Epoxide Hydrolase 1
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2016 (English)In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 14, no 24, p. 5639-5651Article in journal (Refereed) Published
Abstract [en]

Potato epoxide hydrolase 1 (StEH1) is a biocatalytically important enzyme that exhibits rich enantio-and regioselectivity in the hydrolysis of chiral epoxide substrates. In particular, StEH1 has been demonstrated to enantioconvergently hydrolyze racemic mixes of styrene oxide (SO) to yield (R)-1-phenylethanediol. This work combines computational, crystallographic and biochemical analyses to understand both the origins of the enantioconvergent behavior of the wild-type enzyme, as well as shifts in activities and substrate binding preferences in an engineered StEH1 variant, R-C1B1, which contains four active site substitutions (W106L, L109Y, V141K and I155V). Our calculations are able to reproduce both the enantio-and regioselectivities of StEH1, and demonstrate a clear link between different substrate binding modes and the corresponding selectivity, with the preferred binding modes being shifted between the wild-type enzyme and the R-C1B1 variant. Additionally, we demonstrate that the observed changes in selectivity and the corresponding enantioconvergent behavior are due to a combination of steric and electrostatic effects that modulate both the accessibility of the different carbon atoms to the nucleophilic side chain of D105, as well as the interactions between the substrate and protein amino acid side chains and active site water molecules. Being able to computationally predict such subtle effects for different substrate enantiomers, as well as to understand their origin and how they are affected by mutations, is an important advance towards the computational design of improved biocatalysts for enantioselective synthesis.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-282015 (URN)10.1039/C6OB00060F (DOI)000378933400042 ()27049844 (PubMedID)
Funder
Swedish National Infrastructure for Computing (SNIC), 25/2-10EU, European Research Council, 306474;283570Swedish Research Council, 621-2011-6055Carl Tryggers foundation , CTS13:104
Available from: 2016-04-01 Created: 2016-04-01 Last updated: 2017-11-30Bibliographically approved
Barrozo, A., Duarte, F., Bauer, P., Carvalho, A. T. P. & Kamerlin, S. C. L. (2015). Cooperative Electrostatic Interactions Drive Functional Evolution in the Alkaline Phosphatase Superfamily. Journal of the American Chemical Society, 137(28), 9061-9076
Open this publication in new window or tab >>Cooperative Electrostatic Interactions Drive Functional Evolution in the Alkaline Phosphatase Superfamily
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2015 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 137, no 28, p. 9061-9076Article in journal (Refereed) Published
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.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-260856 (URN)10.1021/jacs.5b03945 (DOI)000358556200033 ()26091851 (PubMedID)
Funder
Swedish Research Council, 2010-5026EU, FP7, Seventh Framework Programme, 306474Swedish National Infrastructure for Computing (SNIC), 25/2-10
Note

De 2 första författarna delar förstaförfattarskapet.

Available from: 2015-08-26 Created: 2015-08-25 Last updated: 2017-12-04Bibliographically approved
Amrein, B. A., Bauer, P., Duarte, F., Janfalk Carlsson, Å., Naworyta, A., Mowbray, S. L., . . . Kamerlin, S. C. L. (2015). Expanding the catalytic triad in epoxide hydrolases and related enzymes. ACS Catalysis, 5(10), 5702-5713
Open this publication in new window or tab >>Expanding the catalytic triad in epoxide hydrolases and related enzymes
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2015 (English)In: ACS Catalysis, ISSN 2155-5435, E-ISSN 2155-5435, Vol. 5, no 10, p. 5702-5713Article in journal (Refereed) Published
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.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-260232 (URN)10.1021/acscatal.5b01639 (DOI)000362391500006 ()
Funder
EU, FP7, Seventh Framework Programme, 306474Swedish Research Council, 621-2011-6055, 621-2010-5145Swedish National Infrastructure for Computing (SNIC), 2015/16-12
Available from: 2015-08-18 Created: 2015-08-18 Last updated: 2017-12-04Bibliographically approved
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