Paul Bauer

Force Field Independent Metal Parameters Using a Nonbonded Dummy Model

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 I 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.

Cooperative Electrostatic Interactions Drive Functional Evolution in the Alkaline Phosphatase Superfamily

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.

Empirical valence bond simulations of the hydride transfer step in the monoamine oxidase B catalyzed metabolism of dopamine: EVB Simulations of Hydride Transfer in MAO B

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. Proteins 2014; 82:3347–3355.

Obtaining optical purity for product diols in enzyme-catalyzed epoxide hydrolysis: contributions from changes in both enantio- and regioselectivity.

Enzyme variants of the plant epoxide hydrolase StEH1 displaying improved stereoselectivities in the catalyzed hydrolysis of (2,3-epoxypropyl)benzene were generated by directed evolution. The evolution was driven by iterative saturation mutagenesis in combination with enzyme activity screenings where product chirality was the decisive selection criterion. Analysis of the underlying causes of the increased diol product ratios revealed two major contributing factors: increased enantioselectivity for the corresponding epoxide enantiomer(s) and, in some cases, a concomitant change in regioselectivity in the catalyzed epoxide ring-opening half-reaction. Thus, variant enzymes that catalyzed the hydrolysis of racemic (2,3-epoxypropyl)benzene into the R-diol product in an enantioconvergent manner were isolated.

Energetic Tuning by tRNA Modifications Ensures Correct Decoding of Isoleucine and Methionine on the Ribosome

Chemical modifications of tRNAs are critical for accurate translation of the genetic code on the ribosome. The discrimination between isoleucine (AUA) and methionine (AUG) codons depends on such modifications of the wobble position in isoleucine tRNA anticodon loops, in all kingdoms of life. Bacteria and archaea employ functionally similar lysine- and agmatine-conjugated cytidine derivatives to ensure decoding fidelity, but the thermodynamics underlying codon discrimination remains unknown. Here, we report structure-based computer simulations that quantitatively reveal the energetics of this decoding strategy in archaea. The results further show that the agmatidine modification confers tRNA specificity primarily by desolvation of the incorrect codon in the non-cognate complex. Tautomerism is found to play no significant role in this decoding system as the usual amino form of the modified tRNA is by far the most stable.

Laboratory evolved enzymes provide snapshots of the development of enantioconvergence in enzyme-catalyzed epoxide hydrolysis

Engineered enzyme variants of potato epoxide hydrolase (StEH1) display varying degrees of enrichment of (2R)‐3‐phenylpropane‐1,2‐diol from racemic benzyloxirane. Curiously, the observed increase in the enantiomeric excess of the (R)‐diol is not only a consequence of changes in enantioselectivity for the preferred epoxide enantiomer, but also to changes in the regioselectivity of the epoxide ring opening of (S)‐benzyloxirane. In order to probe the structural origin of these differences in substrate selectivity and catalytic regiopreference, we solved the crystal structures for the evolved StEH1 variants. We used these structures as a starting point for molecular docking studies of the epoxide enantiomers into the respective active sites. Interestingly, despite the simplicity of our docking analysis, the apparent preferred binding modes appear to rationalize the experimentally determined regioselectivities. The analysis also identifies an active site residue (F33) as a potentially important interaction partner, a role that could explain the high conservation of this residue during evolution. Overall, our experimental, structural, and computational studies provide snapshots into the evolution of enantioconvergence in StEH1‐catalyzed epoxide hydrolysis.

The evolution of multiple active site configurations in a designed enzyme

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.Generation and iterative optimization of designed enzymes can provide valuable insights for a more efficient catalysis. Here the authors have followed the iterative improvement of a designed Kemp eliminase and show that remote point mutations could remodel the designed active site via substantial conformational reorganization.

Empirical Valence Bond Simulations Suggest a Direct Hydride Transfer Mechanism for Human Diamine Oxidase

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 substrate–Schiff base to the product–Schiff base, which includes the rate-limiting cleavage of the Cα–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 Cα–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.