246 research outputs found
Unpicking the Cause of Stereoselectivity in Actinorhodin Ketoreductase Variants with Atomistic Simulations
No full textKetoreductase enzymes (KRs) with a high degree of regio- and stereoselectivity are useful biocatalysts for the production of small, specific chiral alcohols from achiral ketones. Actinorhodin KR (actKR), part of a type II polyketide synthase involved in the biosynthesis of the antibiotic actinorhodin, can also turn over small ketones. In vitro studies assessing stereocontrol in actKR have found that, in the "reverse" direction, the wild-type (WT) enzyme's mild preference for S-α-tetralol is enhanced by certain mutations (e.g., P94L) and entirely reversed by others (e.g., V151L) in favor of R-α-tetralol. Here, we employ computationally cost-effective atomistic simulations to rationalize these trends in WT, P94L, and V151L actKR using trans-1-decalone (1) as the model substrate. Three potential factors (FI-FIII) are investigated: frequency of pro-R vs pro-S reactive poses (FI) is assessed with classical molecular dynamics (MD), binding affinity of pro-R vs pro-S orientations (FII) is compared using the binding free energy method MM/PBSA, and differences in reaction barriers toward trans-1-decalol (FIII) are assessed by hybrid semiempirical quantum/classical (QM/MM) MD simulations with umbrella sampling, benchmarked with density functional theory. No single factor is found to dominate stereocontrol: FI largely determines the selectivity of V151L actKR, whereas FIII is more dominant in the case of P94L. It is also found that formation of S-trans-1-decalol or R-trans-1-decalol mainly arises from the reduction of the trans-1-decalone enantiomers (4aS,8aR)-1 or (4aR,8aS)-1, respectively. Our work highlights the complexity of enzyme stereoselectivity as well as the usefulness of atomistic simulations to aid the design of stereoselective biocatalysts.</p
Selected simulation input and output for patchoulol synthase and germacradien-11-ol synthase
No full textFiles related to the biomolecular simulations in the following publication:
Active site loop engineering abolishes water capture in hydroxylating sesquiterpene synthases
Prabhakar L. Srivastava,[a] Sam T. Johns,[b] Rebecca Walters,[b] David J. Miller,[a] Rudolf K. Allemann*[a] and Marc W. Van der Kamp*[b]
[a]School of Chemistry, Cardiff University, Main Building, Park Place, Cardiff CF10 3AT, United Kingdom
[b]School of Biochemistry, University of Bristol, University Walk, Bristol BS8 1TD, United Kingdom
See the README.docx document for details
MM parameters for covalent adducts of Clavulanate degradation by beta-lactamases
No full textThis dataset contains parameters for the acyl-enzyme complexes simulated in the following work:Multiscale simulations establish clavulanate inhibition efficiency and the responsible enzyme complex in class A β-lactamases. [1]Rubén A. Fritz, Jans H. Alzate-Morales, James Spencer, Adrian J. Mulholland and Marc W. Van der Kamp. MM parameters for the following covalent complexes resulting from inhibition with clavulanate are present: AEC - acyl-enzyme complex of clavulanate (incl. Ser70) Files: AEC.off, AEC.frcmod Residue name: AEC TEDC - decarboxylated trans-enamine complex (incl. Ser70) that is the result of opening of the five-membered ring (leading to an imine intermediate) and subsequent rearrangement [2,3] Files: TEDC.off, TEDC.frcmod Residue name: TDCADEC - aldehyde complex (incl. Ser70) that is the result of consecutive reactions of the cis-enamine [2,3] Files: ADEC.off, ADEC.frcmod Residue name: ADC Parameterisation procedure: Initial Amber ff14SB force field parameters for the acylated residue in the models (adducts) were obtained from the RED Server (partial charges from HF/6-31G(d,p) RESP-fitting and atom types). For missing parameters, chemically equivalent parameters from the GAFF force field were used. [1] Fritz RA, Alzate-Morales JH, Spencer J, Mulholland AJ and Van der Kamp MW. Biochemistry (2018). Under review.[2] Drawz, S. M., and Bonomo, R. a. (2010) Three decades of β-lactamase inhibitors. Clin. Microbiol. Rev. 23, 160–201. [3] Helfand, M. S., Totir, M. A., Carey, M. P., Hujer, A. M., Bonomo, R. A., and Carey, P. R. (2003) Following the Reactions of Mechanism-Based Inhibitors with β-Lactamase by Raman Crystallography. Biochemistry 42, 13386–13392.</div
Carbepenem acyl-enzyme parameters (for beta-lactamase simulations): Meropenem
No full textAcyl-enzyme parameters used in the following publication:"QM/MM simulations as an assay for carbapenemase activity in class A β-lactamases"Chudyk EI, Limb MAL,
Jones C, Spencer J, Van der Kamp MW,
Mulholland AJ. 2014. Chem Commun 50,
14736DOI: 10.1039/c4cc06495jAcyl-enzyme of Meropenem (incl. Ser70) Files: MER.off, MER.frcmodResidue name: MERAtom names are labeled in: MER_atom_names.pngParameterisation procedure: Initial Amber ff14SB force field parameters for the acylated Ser-70 residue (with caps on either side) were obtained from the RED Server (partial charges from HF/6-31G(d,p) RESP-fitting and atom types). For missing parameters, chemically equivalent parameters from the GAFF force field were used.</div
Path to Actinorhodin:Regio- and Stereoselective Ketone Reduction by a Type II Polyketide Ketoreductase Revealed in Atomistic Detail
No full textABSTRACT: In type II polyketide synthases (PKSs), which typically biosynthesize several antibiotic and antitumor compounds, the substrate is a growing polyketide chain, shuttled between individual PKS enzymes whilst covalently tethered to an acyl carrier protein (ACP): this requires the ACP interacting with a series of different enzymes in succession. During biosynthesis of the antibiotic actinorhodin, produced by Streptomyces cœlicolor, one such key binding event is between an ACP carrying a 16-carbon octaketide chain (actACP) and a ketoreductase (actKR). Once the octaketide is bound inside actKR, it is likely cyclized between C7 and C12 and regioselective reduction of the ketone at C9 occurs: how these elegant chemical and conformational changes are controlled is not yet known. Here, we perform protein-protein docking, protein NMR, and extensive molecular dynamics simulations to reveal a probable mode of association between actACP and actKR; we obtain and analyze a detailed model of the C7-C12-cyclized octaketide within the actKR active site; and confirm this model through multiscale (QM/MM) reaction simulations of the key ketoreduction step. Molecular dynamics simulations show that the most thermodynamically stable cyclized octaketide isomer (7R,12R) also gives rise to the most reaction competent conformations for ketoreduction. Subsequent reaction simulations show that ketoreduction is stereoselective as well as regioselective, resulting in an S-alcohol. Our simulations further indicate several conserved residues that may be involved in selectivity of C7- 12 cyclization and C9 ketoreduction. Detailed insights obtained on ACP-based substrate presentation in type II PKSs can help design ACP-ketoreductase systems with altered regio- or stereoselectivity
Simulation of Functional Motions in Enzymes
No full textData related to: "Structure and function in homodimeric enzymes: simulations of cooperative and independent functional motions". Wells SA, Van der Kamp MW, Mulholland AJ. PLOS ONE, 2015. Results from two different simulation methods, normal-mode biased geometric simulations of flexible motion and conventional molecular dynamics, as applied to two different homodimeric enzymes, the DcpS scavanger decapping enzyme and citrate synthase
Understanding Complex Mechanisms of Enzyme Reactivity:The Case of Limonene-1,2-Epoxide Hydrolases
No full textLimonene-1,2-epoxide hydrolases (LEHs), a subset of the epoxide hydrolase family, present interesting opportunities for the mild, regio- and stereo- selective hydrolysis of epoxide substrates. However, moderate enantioselectivity for non-natural ligands, combined with narrow substrate specificity, has so far limited the use of LEHs as general biocatalytic tools. A detailed molecular understanding of the structural and dynamic determinants of activity may complement directed evolution approaches to expand the range of applicability of these enzymes. Herein, we have combined quantum mechanics/molecular mechanics (QM/MM) free energy calculations for the reaction with MD simulations of the enzyme internal dynamics, and the calculation of binding affinities (using the WaterSwap method) for various representatives of the enzyme conformational ensemble, to show that the presence of natural or non-natural substrates differentially modulates the dynamic and catalytic behavior of LEH. The cross-talk between the protein and the ligands favors the selection of specific substrate-dependent interactions in the binding site, priming reactive complexes to select different preferential reaction pathways. The knowledge gained via our combined approach provides a molecular rationale for LEH substrate preferences. The comprehensive strategy we present here is general and broadly applicable to other cases of enzyme-substrate selectivity and reactivity.</p
Comparison of different quantum mechanical/molecular mechanics boundary treatments in the reaction of the hepatitis C virus NS3 protease with the NS5A/5B substrate
No full textEpistasis Arises from Shifting the Rate-Limiting Step during Enzyme Evolution of a β-lactamase
No full textData related to: "Epistasis Arises from Shifting the Rate-Limiting Step during Enzyme Evolution of a β-lactamase". Christopher Fröhlich, H. Adrian Bunzel, Karol Buda, Adrian J. Mulholland, Marc W. van der Kamp, Pål J. Johnsen, Hanna-Kirsti S. Leiros, Nobuhiko Tokuriki Nature Catalysis 2023. This repository contains Jupyter notebooks and input files required to replicate the MD simulations and analyses of the OXA-48 variants, as well as trajectories and MD snapshots. The repository furthermore contains PDB structures of all ensemble refinements presented in this work
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