Maciej Szaleniec

BN/CC Isosteric Compounds as Enzyme Inhibitors: N- and B-Ethyl-1,2-azaborine Inhibit Ethylbenzene Hydroxylation as Nonconvertible Substrate Analogues

Good substrate gone bad! BN/CC isosterism of ethylbenzene leads to N-ethyl-1,2-azaborine and B-ethyl-1,2-azaborine. In contrast to ethylbenzene, which is the substrate for ethylbenzene dehydrogenase (EbDH), N-ethyl-1,2-azaborine (see scheme; Fc=Ferricenium tetrafluoroborate) and B-ethyl-1,2-azaborine are strong inhibitors of EbDH. Thus, the changes provided by BN/CC isosterism can lead to new biochemical reactivity.

Ab Inito Modeling of Ethylbenzene Dehydrogenase Reaction Mechanism

Density functional theory calculations were performed to study the mechanism of ethylbenzene oxidation by ethylbenzene dehydrogenase (EBDH). EBDH is a bacterial molybdopterin enzyme capable of stereospecific anaerobic hydroxylation of alkylaromatic compounds to secondary alcohols. It is a key biocatalyst in the metabolism of ethylbenzene-degrading bacteria such as Aromatoleum aromaticum , which converts ethylbenzene to (S)-1-phenylethanol. The recently determined EBDH structure enabled the theoretical description of the ethylbenzene oxidation mechanism. In this work, theoretical calculations and kinetic isotopic experiments were conducted and combined in order to elucidate the reaction mechanism. We considered three aspects: (i) Does the reaction concur with one two-electron or two one-electron transfers? (ii) Is the active site His192 important for the reaction and what is its protonation state? (iii) What catalytic consequences have different possible arrangements of the molybdopterin ligand? The most important outcome of the calculations is that mechanisms involving two one-electron transfers and a radical-type intermediate have lower energy barriers than the corresponding two-electron transfer mechanisms and are, therefore, more plausible. The mechanism involves two transition states: radical-type TS1 associated with the C-H bond cleavage, and carbocation-type TS2 associated with the transfer of the second electron and OH rebound. Using models with protonated and nonprotonated His 192, we conclude that this amino acid takes part in the mechanism. However, as both models yielded plausible reaction pathways, its protonation state cannot be easily predicted. Qualitative agreement was reached between the calculated kinetic isotope effects (KIE) obtained for radical TS1 and the KIE measured experimentally at optimum pH, but we observed a very strong pH dependence of KIE throughout the investigated pH range (3.1 for pH 6, 5.9 for pH 7, up to 10.5 at pH 8.). This may be explained by assuming a gradual shift of the rate-determining step from TS1 associated with high KIE to TS2 associated with low KIE with lowered pH and an increasing contribution of proton/deuteron tunneling associated with high pH. Finally, models were calculated with different signs of the conformational twist of the pterin ligands, yielding only slightly different energy profiles of the reaction pathways.

How to select an optimal neural model of chemical reactivity

The paper aims at methodological studies on selection of optimal neural network that performs modeling of chemical reactivity of a given group of chemical compounds. The problem (prediction of biological activity in enzymatic reaction catalyzed by ethylbenzene dehydrogenase) is taken as a case study for assessment of various types of neural networks. The main goal of the study is to select the best type of the network, optimal dimension of the layers, proper parameters of the network as well as the optimal form of data representation for purpose of neural-based modeling of complex empirical data. Various approaches (linear networks, regression and classification multiple layer perceptrons, generalized regression neural networks) are compared and tested.

Asymmetric reduction of ketones and β-keto esters by (S)-1-phenylethanol dehydrogenase from denitrifying bacterium Aromatoleum aromaticum

Enzyme-catalyzed enantioselective reductions of ketones and keto esters have become popular for the production of homochiral building blocks which are valuable synthons for the preparation of biologically active compounds at industrial scale. Among many kinds of biocatalysts, dehydrogenases/reductases from various microorganisms have been used to prepare optically pure enantiomers from carbonyl compounds. (S)-1-phenylethanol dehydrogenase (PEDH) was found in the denitrifying bacterium Aromatoleum aromaticum (strain EbN1) and belongs to the short-chain dehydrogenase/reductase family. It catalyzes the stereospecific oxidation of (S)-1-phenylethanol to acetophenone during anaerobic ethylbenzene mineralization, but also the reverse reaction, i.e., NADH-dependent enantioselective reduction of acetophenone to (S)-1-phenylethanol. In this work, we present the application of PEDH for asymmetric reduction of 42 prochiral ketones and 11 β-keto esters to enantiopure secondary alcohols. The high enantioselectivity of the reaction is explained by docking experiments and analysis of the interaction and binding energies of the theoretical enzyme-substrate complexes leading to the respective (S)- or (R)-alcohols. The conversions were carried out in a batch reactor using Escherichia coli cells with heterologously produced PEDH as whole-cell catalysts and isopropanol as reaction solvent and cosubstrate for NADH recovery. Ketones were converted to the respective secondary alcohols with excellent enantiomeric excesses and high productivities. Moreover, the progress of product formation was studied for nine para-substituted acetophenone derivatives and described by neural network models, which allow to predict reactor behavior and provides insight on enzyme reactivity. Finally, equilibrium constants for conversion of these substrates were derived from the progress curves of the reactions. The obtained values matched very well with theoretical predictions.

Ethylbenzene Dehydrogenase and Related Molybdenum Enzymes Involved in Oxygen-Independent Alkyl Chain Hydroxylation.

Ethylbenzene dehydrogenase initiates the anaerobic bacterial degradation of ethylbenzene and propylbenzene. Although the enzyme is currently only known from a few closely related denitrifying bacterial strains affiliated to the Rhodocyclaceae, it clearly marks a universally occurring mechanism used for attacking recalcitrant substrates in the absence of oxygen. Ethylbenzene dehydrogenase belongs to subfamily 2 of the DMSO reductase-type molybdenum enzymes together with paralogous enzymes involved in the oxygen-independent hydroxylation of p-cymene, the isoprenoid side chains of sterols and even possibly n-alkanes; the subfamily also extends to dimethylsulfide dehydrogenases, selenite, chlorate and perchlorate reductases and, most significantly, dissimilatory nitrate reductases. The biochemical, spectroscopic and structural properties of the oxygen-independent hydroxylases among these enzymes are summarized and compared. All of them consist of three subunits, contain a molybdenum-bis-molybdopterin guanine dinucleotide cofactor, five Fe-S clusters and a heme b cofactor of unusual ligation, and are localized in the periplasmic space as soluble enzymes. In the case of ethylbenzene dehydrogenase, it has been determined that the heme b cofactor has a rather high redox potential, which may also be inferred for the paralogous hydroxylases. The known structure of ethylbenzene dehydrogenase allowed the calculation of detailed models of the reaction mechanism based on the density function theory as well as QM-MM (quantum mechanics - molecular mechanics) methods, which yield predictions of mechanistic properties such as kinetic isotope effects that appeared consistent with experimental data.

Mechanistic basis for the Enantioselectivity of the Anaerobic Hydroxylation of Alkylaromatic compounds by Ethylbenzene Dehydrogenase.

The enantioselectivity of reactions catalyzed by ethylbenzene dehydrogenase, a molybdenum enzyme that catalyzes the oxygen-independent hydroxylation of many alkylaromatic and alkylheterocyclic compounds to secondary alcohols, was studied by chiral chromatography and theoretical modeling. Chromatographic analyses of 22 substrates revealed that this enzyme exhibits remarkably high reaction enantioselectivity toward (S)-secondary alcohols (18 substrates converted with >99% ee). Theoretical QM:MM modeling was used to elucidate the structure of the catalytically active form of the enzyme and to study the reaction mechanism and factors determining its high degree of enantioselectivity. This analysis showed that the enzyme imposes strong stereoselectivity on the reaction by discriminating the hydrogen atom abstracted from the substrate. Activation of the pro(S) hydrogen atom was calculated to be 500 times faster than of the pro(R) hydrogen atom. The actual hydroxylation step (i.e., hydroxyl group rebound reaction to a carbocation intermediate) does not appear to be enantioselective enough to explain the experimental data (the calculated rate ratios were in the range of only 2-50 for pro(S): pro(R)-oriented OH rebound).

Substrate and Inhibitor Spectra of Ethylbenzene Dehydrogenase: Perspectives on Application Potential and Catalytic Mechanism

ABSTRACT Ethylbenzene dehydrogenase (EbDH) catalyzes the initial step in anaerobic degradation of ethylbenzene in denitrifying bacteria, namely, the oxygen-independent hydroxylation of ethylbenzene to (S)-1-phenylethanol. In our study we investigate the kinetic properties of 46 substrate analogs acting as substrates or inhibitors of the enzyme. The apparent kinetic parameters of these compounds give important insights into the function of the enzyme and are consistent with the predicted catalytic mechanism based on a quantum chemical calculation model. In particular, the existence of the proposed substrate-derived radical and carbocation intermediates is substantiated by the formation of alternative dehydrogenated and hydroxylated products from some substrates, which can be regarded as mechanistic models. In addition, these results also show the surprisingly high diversity of EbDH in hydroxylating different kinds of alkylaromatic and heterocyclic compounds to the respective alcohols. This may lead to attractive industrial applications of ethylbenzene dehydrogenase for a new process of producing alcohols via hydroxylation of the corresponding aromatic hydrocarbons rather than the customary procedure of reducing the corresponding ketones.

Structure and Function of Benzylsuccinate Synthase and Related Fumarate-Adding Glycyl Radical Enzymes

The pathway of anaerobic toluene degradation is initiated by a remarkable radical-type enantiospecific addition of the chemically inert methyl group to the double bond of a fumarate cosubstrate to yield (R)-benzylsuccinate as the first intermediate, as catalyzed by the glycyl radical enzyme benzylsuccinate synthase. In recent years, it has become clear that benzylsuccinate synthase is the prototype enzyme of a much larger family of fumarate-adding enzymes, which play important roles in the anaerobic metabolism of further aromatic and even aliphatic hydrocarbons. We present an overview on the biochemical properties of benzylsuccinate synthase, as well as its recently solved structure, and present the results of an initial structure-based modeling study on the reaction mechanism. Moreover, we compare the structure of benzylsuccinate synthase with those predicted for different clades of fumarate-adding enzymes, in particular the paralogous enzymes converting p-cresol, 2-methylnaphthalene or n-alkanes.

Artificial neural network modelling of the results of tympanoplasty in chronic suppurative otitis media patients

The application of computer modelling for medical purposes, although challenging, is a promising pathway for further development in the medical sciences. We present predictive neural and k-nearest neighbour (k-NN) models for hearing improvements after middle ear surgery for chronic otitis media. The studied data set comprised 150 patients characterised by the set of input variables: age, gender, preoperative audiometric results, ear pathology and details of the surgical procedure. The predicted (output) variable was the postoperative hearing threshold. The best neural models developed in this study achieved 84% correct predictions for the test data set while the k-NN model produced only 75.8% correct predictions.

Effects of metal cations on betanin stability in aqueous-organic solutions

An effect of metal cations on betanin stability was investigated in aqueous and organic-aqueous solutions. The presence of organic solvents (methanol, ethanol, and acetonitrile) affects substantially the pigments decomposition in acidic media induced by metal cations whose degrading action in such media is significantly higher than in aqueous solutions. The influence of Cu2+ on the stability was studied by spectrophotometry in more detailed manner, because of its ability to form complexes with betanin. The possibility of a complex formation between betanin and Ni2+ was also stated at pH 7–8; its relatively high stability in aqueous samples was observed. A presence of numerous products of betanin decomposition was detected in the wavelength range 380–500 nm in spectra obtained for most of metal cations investigated, especially for higher concentrations of the organic solvents.