Development of electrochemical biosensors based on sol-gel enzyme encapsulation and protective polymer membranes

Protective polymer coatings have been used to enhance the retention of enzymes in sol-gel films as immobilisation phases in electrochemical biosensors. Carbon film electrodes were electrochemically modified with poly(neutral red) (PNR). These electrodes were coated with oxysilane sol-gels incorporating glucose oxidase and an outer coating of carboxylated PVC (CPVC) or polyurethane (PU), with and without Aliquat-336 or isopropyl myristate (IPM) plasticizer, was applied. The biosensors were characterised electrochemically using cyclic voltammetry and amperometry, electrochemical impedance spectroscopy and scanning electron microscopy. Impedance spectra showed that the electrode surface is most active when the sol-gel–GOx layer is not covered with a membrane. However, membranes without plasticizer extend the lifetime of the biosensor to more than 2 months when PU is used as an outer membrane. The linear range of the biosensors was found to be 0.05–0.50 mM of glucose and the biosensor with PU outer membrane exhibited higher sensitivity (ca.117 nA mM⁻¹) in the region of linear response than that with CPVC. The biosensors were applied to glucose measurement in natural samples of commercial orange juice.     

In Silico Analysis of P450s and Their Role in Secondary Metabolism in the Bacterial Class Gammaproteobacteria

The impact of lifestyle on shaping the genome content of an organism is a well-known phenomenon and cytochrome P450 enzymes (CYPs/P450s), heme-thiolate proteins that are ubiquitously present in organisms, are no exception. Recent studies focusing on a few bacterial species such as Streptomyces, Mycobacterium, Cyanobacteria and Firmicutes revealed that the impact of lifestyle affected the P450 repertoire in these species. However, this phenomenon needs to be understood in other bacterial species. We therefore performed genome data mining, annotation, phylogenetic analysis of P450s and their role in secondary metabolism in the bacterial class Gammaproteobacteria. Genome-wide data mining for P450s in 1261 Gammaproteobacterial species belonging to 161 genera revealed that only 169 species belonging to 41 genera have P450s. A total of 277 P450s found in 169 species grouped into 84 P450 families and 105 P450 subfamilies, where 38 new P450 families were found. Only 18% of P450s were found to be involved in secondary metabolism in Gammaproteobacterial species, as observed in Firmicutes as well. The pathogenic or commensal lifestyle of Gammaproteobacterial species influences them to such an extent that they have the lowest number of P450s compared to other bacterial species, indicating the impact of lifestyle on shaping the P450 repertoire. This study is the first report on comprehensive analysis of P450s in Gammaproteobacteria.     

Reconstitution of the Cytb5–CytP450 Complex in Nanodiscs for Structural Studies using NMR Spectroscopy

Cytochrome P450s (P450s) are a superfamily of enzymes responsible for the catalysis of a wide range of substrates. Dynamic interactions between full‐length membrane‐bound P450 and its redox partner cytochrome b₅ (cytb₅) have been found to be important for the enzymatic activity of P450. However, the stability of the circa 70 kDa membrane‐bound complex in model membranes renders high‐resolution structural NMR studies particularly difficult. To overcome these challenges, reconstitution of the P450–cytb₅ complex in peptide‐based nanodiscs, containing no detergents, has been demonstrated, which are characterized by size exclusion chromatography and NMR spectroscopy. In addition, NMR experiments are used to identify the binding interface of the P450–cytb₅ complex in the nanodisc. This is the first successful demonstration of a protein–protein complex in a nanodisc using NMR structural studies and should be useful to obtain valuable structural information on membrane‐bound protein complexes.     

Cytochrome p450 compound I

Cytochrome P450 enzymes (P450s) comprise a large class of enzymes that effect numerous oxidations in nature. The active oxidants in P450s are thought to be iron(IV)-oxo porphyrin radical cations termed Compounds I, and these intermediates have been sought since the discovery of P450s 40 years ago. We report formation of the Compound I derivative of a P450 enzyme by laser flash photolysis oxidation of the corresponding Compound II species, an iron(IV)-oxo neutral porphyrin intermediate. The Compound II derivative in turn was produced by oxidation of the P450 with peroxynitrite, which effected a net one-electron, oxo-transfer reaction to the iron(III) atom of the resting enzyme. For the P450 studied in this work, CYP119 from the thermophile Sulfolobus solfactaricus, the P450 Compound II derivative was stable for seconds at ambient temperature, and the Compound I transient decayed with a lifetime of ca. 200 ms.     

Calculating physical properties of organic compounds for environmental modeling from molecular structure

Mathematical models for predicting the transport and fate of pollutants in the environment require reactivity parameter values – that is the value of the physical and chemical constants that govern reactivity. Although empirical structure–activity relationships have been developed that allow estimation of some constants, such relationships are generally valid only within limited families of chemicals. The computer program, SPARC, uses computational algorithms based on fundamental chemical structure theory to estimate a large number of chemical reactivity parameters and physical properties for a wide range of organic molecules strictly from molecular structure. Resonance models were developed and calibrated using measured light absorption spectra, whereas electrostatic interaction models were developed using measured ionization pK_as in water. Solvation models (i.e., dispersion, induction, H-bonding, etc.) have been developed using various measured physical properties data. At the present time, SPARC’s physical property models can predict vapor pressure and heat of vaporization (as a function of temperature), boiling point (as a function of pressure), diffusion coefficient (as a function of pressure and temperature), activity coefficient, solubility, partition coefficient and chromatographic retention time as a function of solvent and temperature. This prediction capability crosses chemical family boundaries to cover a broad range of organic compounds.     

A Panel of Cytochrome P450 BM3 Variants to Produce Drug Metabolites and Diversify Lead Compounds

Herein we demonstrate that a small panel of variants of cytochrome P450 BM3 from Bacillus megaterium covers the breadth of reactivity of human P450s by producing 12 of 13 mammalian metabolites for two marketed drugs, verapamil and astemizole, and one research compound. The most active enzymes support preparation of individual metabolites for preclinical bioactivity and toxicology evaluations. Underscoring their potential utility in drug lead diversification, engineered P450 BM3 variants also produce novel metabolites by catalyzing reactions at carbon centers beyond those targeted by animal and human P450s. Production of a specific metabolite can be improved by directed evolution of the enzyme catalyst. Some variants are more active on the more hydrophobic parent drug than on its metabolites, which limits production of multiply‐hydroxylated species, a preference that appears to depend on the evolutionary history of the P450 variant.     

P450 fingerprinting method for rapid discovery of terpene hydroxylating P450 catalysts with diversified regioselectivity

Engineered P450 enzymes constitute attractive catalysts for the selective oxidation of unactivated C-H bonds in complex molecules. A current bottleneck in the use of P450 catalysis for chemical synthesis is the time and effort required to identify the P450 variant(s) with the desired level of activity and selectivity. In this report, we describe a method to map the active site configuration of engineered P450 variants in high throughput using a set of semisynthetic chromogenic probes. Through analysis of the resulting 'fingerprints', reliable predictions can be made regarding the reactivity of these enzymes toward complex substrates structurally related to the fingerprint probes. In addition, fingerprint analysis offers a convenient and time-effective means to assess the regioselectivity properties of the fingerprinted P450s. The described approach can represent a valuable tool to expedite the discovery of P450 oxidation catalysts for the functionalization of relevant natural products such as members of the terpene family.     

Fourier transform infrared spectroscopy as a tool to study structural properties of cytochromes P450 (CYPs)

Cytochrome P450 proteins (CYPs) are a big class of heme proteins which are involved in various metabolic processes of living organisms. CYPs are the terminal catalytically active components of monooxygenase systems where the substrate binds and is hydroxylated. In order to be functionally competent, the protein structures of CYPs possess specific properties that must be explored in order to understand structure–function relationships and mechanistic aspects. Fourier transform infrared spectroscopy (FTIR) is one tool that is used to study these structural properties. The application of FTIR spectroscopy to the secondary structures of CYP proteins, protein unfolding, protein–protein interactions and the structure and dynamics of the CYP heme pocket is reviewed. A comparison with other thiolate heme proteins (nitric oxide synthase and chloroperoxidase) is also included. Figure The protein secondary structure, protein unfolding, redox-partner protein–protein interaction, structural changes induced by the reduction of the heme iron, and the structure and dynamics of the active site of cytochromes P450 (CYP) can be studied using Fourier transform infrared spectroscopy (FTIR). FTIR spectroscopy is a good approach for gaining a deeper insight into structure–function relationships in CYPs. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Structural characterization of agmatine at physiological conditions

The present work aims at determining the structure–activity relationships (SAR’s) which rule the biological function of agmatine (4-(aminobutyl)guanidinium, AGM), a biogenic amine produced by decarboxylation of arginine. Its structural preferences, both as an isolated molecule and in aqueous solution (namely at physiological conditions) were ascertained, by vibrational (Raman) spectroscopy coupled to theoretical (density functional) calculations. An evaluation of mitochondrial functions (membrane potential (ΔΨ), mitochondrial swelling, and cytochrome c release) in rat liver mitochondria (RLM) was also carried out. The results thus obtained, coupled to the conformational analysis performed for the distinct polyamine protonation states, allowed to individualize the agmatine structures which interact with the mitochondrial site responsible for its transport and for the protection against mitochondrial permeability transition (MPT) induction, as well as to gain information on the specific mechanisms involved.     

细胞色素p450的结构与催化机理

细胞色素P450酶是广泛存在的含亚铁血红素单加氧酶, 参与甾类激素的合成、脂溶性维生素代谢、多不饱和脂肪酸转换为生物活性分子, 以及致癌作用和药物代谢. 综述了细胞色素p450结构与功能的关系, 特别是细胞色素P450活性位点经历大幅度开/关运动结合底物和释放产物以及电子迁移途径.     

Immobilization of a Bacterial Cytochrome P450 Monooxygenase System on a Solid Support

Bacterial cytochrome P450s (P450s), which catalyze regio‐ and stereoselective oxidations of hydrocarbons with high turnover rates, are attractive biocatalysts for fine chemical production. Enzyme immobilization is needed for cost‐effective industrial manufacturing. However, immobilization of P450s is difficult because electron‐transfer proteins are involved in catalysis and anchoring these can prevent them from functioning as shuttle molecules for carrying electrons. We studied a heterotrimeric protein‐mediated co‐immobilization of a bacterial P450, and its electron‐transfer protein and reductase. Fusion with subunits of a heterotrimeric Sulfolobus solfataricus proliferating cell nuclear antigen (PCNA) enabled immobilization of the three proteins on a solid support. The co‐immobilized enzymes catalyzed monooxygenation because the electron‐transfer protein fused to PCNA via a single peptide linker retained its electron‐transport function.     

Development and validation of a LC-MS/MS method based on a new 96-well Hybrid-SPE<Superscript>™</Superscript>-precipitation technique for quantification of CYP450 substrates/metabolites in rat plasma

A rapid and selective high-throughput HESI-LC-MS/MS method for determining eight cytochrome P450 probe drugs in one-step extraction and single run was developed and validated. The four specific probe substrates midazolam, dextromethorphan, tolbutamide, theophylline and their metabolites 1-hydroxymidazolam, dextrorphan, hydroxyl(methyl)tolbutamide, 1,3-dimethyluric acid, together with the deuterated internal standards, were extracted from rat plasma using a novel 96-well Hybrid-SPE™-precipitation technique. The bioanalytical assay was based on reversed phase liquid chromatography coupled with tandem mass spectrometry in the positive ion mode using selected reaction monitoring for drug (-metabolite) quantification. All analytes were separated simultaneously in a single run that lasted less than 11 min. The intra- and inter-day precisions for all eight substrates/metabolites were 1.62–12.81% and 2.09–13.02%, respectively, and the relative errors (accuracy) for the eight compounds ranged from −9.62% to 7.48% and −13.84% to 8.82%. Hence, the present method provides a robust, fast and reproducible analytical tool for the evaluation of four major drug metabolising cytochrome P450 (3A4, 2C9, 1A2 and 2D6) activities with a cocktail approach in rats to clarify herb–drug interactions. The method can be used as a basic common validated high-throughput analytical assay for in vivo interaction studies.     

Predicting Drug Interaction of Clopidogrel on Microbial Metabolism of Diclofenac

Seven fungal cultures were studied for the metabolism of diclofenac in order to elucidate the nature of enzymes involved in biotransformation, as diclofenac is a specific substrate to cytochrome P450 (CYP) 2C9 isozyme in mammals. The metabolites were identified by high-performance liquid chromatography–diode array detection and liquid chromatography–tandem mass spectroscopy analysis. The study included clopidogrel, a selective inhibitor of CYP2C9 isozyme, to inhibit the metabolism of diclofenac. Two-stage fermentation protocol was used to study the diclofenac metabolism and its inhibition by clopidogrel. Among the cultures studied, four have shown positive indication for drug interaction, since clopidogrel inhibited the metabolism of diclofenac in a dose-dependent manner. The results indicate that microbial cultures possess enzyme systems similar to mammals and they can be used to predict drug interactions in mammalian systems.     

Prostacyclin and Thromboxane Synthase: New Aspects of Hemethiolate Catalysis

The arachidonic acid metabolites thromboxane A₂ and prostacyclin are highly potent regulators of cell physiology. They are both formed by enzymatic rearrangement of the 9,11-epidioxy prostaglandin H₂ catalyzed, however, by thromboxane and prostacyclin synthase, respectively. The two enzymes have been isolated, sequenced, and characterized as hemethiolate (“P450”) enzymes. The different isomerization products can be explained on the same catalytic principle by a different ligation of the heme centers with the two epidioxy oxygens atoms. This requires different conformations for substrate binding at the active site, which is substantiated by the different inhibitors and amino acid sequences of the enzymes. In a hypothesis which has mechanistic principles in common with the P450-monooxygenases and the allene oxide synthases, oxy radicals are formed first and rearrange to carbon radicals. These could then rapidly be converted into carbocations by the ferrylthiolate or iron(III )thiyl structures formed as intermediates.     

Lactose Hydrolysis by β-Galactosidase Covalently Immobilized to Thermally Stable Biopolymers

Lactose has been hydrolyzed using covalently immobilized β-galactosidase on thermally stable carrageenan coated with chitosan (hydrogel). The hydrogel’s mode of interaction was proven by Fourier transform infrared spectroscopy, differential scanning calorimetry (DSC), and Schiff’s base formation. The DSC thermogram proved the formation of a strong polyelectrolyte complex between carrageenan and chitosan followed by glutaraldehyde as they formed one single peak. The modification of carrageenan improved the gel’s thermal stability in solutions from 35 °C to 95 °C. The hydrogel has been proven to be efficient for β-galactosidase immobilization where 11 U/g wet gel was immobilized with 50% enzyme loading capacity. Activity and stability of free and immobilized β-galactosidase towards pH and temperature showed marked shifts in their optimum pH from 4.5–5 to 5–5.5 and temperature from 50 °C to 45–55 °C after immobilization, which reveals higher catalytic activity and reasonable stability at wider pHs and temperatures. The apparent K _m of the immobilized enzyme increased from 13.2 to 125 mM, whereas the V _max increased from 3.2 to 6.6 μmol/min compared to the free enzyme, respectively. The free and immobilized enzymes showed lactose conversion of 87% and 70% at 7 h, respectively. The operational stability showed 97% retention of the enzyme activity after 15 uses, which demonstrates that the covalently immobilized enzyme is unlikely to leach. The new carrier could be suitable for immobilization of other industrial enzymes.     

Synthesis and Transport and Ionophoric Properties of α,ω-Diphosphorylated Azapodands: XII. Membrane Transport of Organic Acids by Diphosphorylated Diamines and Diazapodands

Membrane transport of mono- and polyfunctional carboxylic acids mediated by phosphorylated diamines has been studied. The structure of complexes transferred through the membrane has been discussed; in some cases, a relation has been found between the efficiency of membrane transport of organic acid substrates and the structure of membrane carriers and their complexes with the acids. A correlation between the transmembrane flow and shift of some characteristic IR absorption bands (as well as change of their intensity) of the substrate–carrier complexes in comparison to the corresponding substrates and carriers has been estimated.     

Quantitative analysis of highly homologous proteins: the challenge of assaying the “CYP-ome” by mass spectrometry

Cytochrome P450 enzymes comprise families of highly homologous proteins. These proteins play a pivotal role in oxidative drug metabolism and are important targets in drug discovery research. Proteomics today is a valuable tool for the analysis of proteins. In the past, qualitative analysis of the proteome was the main focus of research, but in the last few years interest in the mathematical modelling of protein networks has been growing and so has the demand on quantitative proteome analysis. As a thorough understanding of cytochrome P450 dependent metabolism is crucial for drug discovery, it is thus not astounding that cytochrome P450 enzymes are a target for quantitative proteomics research. In this article, we review the techniques available for quantitative proteome analysis and to what extent these techniques have been used for the quantification of cytochrome P450 enzymes and give a brief outlook of the techniques that have promising potential for the analysis of these proteins in the future.     

The inhibitory effect of the artificial electron donor system, phenazine methosulfate-ascorbate, on bacterial transport mechanisms.

The artificial electron donor system, phenazine methosulfate (PMS)-ascorbate, inhibited active transort of solutes in Pseudomonas aeruginosa irrespective of whether the active transport systems were shock sensitive or shock resistant. N,N,N',N'-tetramethylphenylenediamine could be substituted for PMS but a higher concentration was required. PMS-ascorbate also inhibited active transport in several other bacterial species with the exception of Escherichia coli and of a nonpigmented strain of Serratia marcescens. PMS-ascorbate previously has been shown to energize active transport in isolated membrane vesicles, even those prepared from the same bacterial species in whose intact cells active transport was inhibited. The apparent Km of glucose active transport in untreated cells of P. aeruginosa was 40 micron while the Km of glucose transport in cells incubated with PMS-ascorbate was 25 mM, and PMS-ascorbate had no effect on efflux of accumulated glucose. These results strongly suggested that facilitated diffusion resulted upon exposure of the cells to PMS- ascorbate. Thus, PMS-ascorbate appeared to have an uncoupler-like effect on cells of P. aeruginosa. The experimental data also pointed out that there are fundamental differences between the response of intact cells and membrane vesicles to exogenous electron donors.     

Radiation-thermal hydrodesulphurization of brown coal

The results of the investigation of radiation-thermal hydrodesulphurization of brown coal in presence of methane under accelerated electrons with a dose rate P=350 Gy/s in the intervals of temperatureT=200–500 °C, absorbed dose up to 60 kGy are presented. It has been established that at the more favourable conditions (T=400–450 °C,D>60 kGy) about 80% of sulphur is removed from the coal and in the solid product sulphur decreased up to 1.0–1.1%. In these conditions the selectivity of desulphurization processes iss=2.6–3.8 and energy consumption towards removal of sulphur isE=0.5–0.6 kW h/kg. The mechanism of selective activation by irradiation for the desulphurization process is discussed.     

Molecular modeling of cytochrome P450 3A4

The three-dimensional structure of human cytochrome P450 3A4 was modeled based on crystallographic coordinates of four bacterial P450s: P450 BM-3, P450cam, P450terp, and P450eryF. The P450 3A4 sequence was aligned to those of the known proteins using a structure-based alignment of P450 BM-3, P450cam, P450terp, and P450eryF. The coordinates of the model were then calculated using a consensus strategy, and the final structure was optimized in the presence of water. The P450 3A4 model resembles P450 BM-3 the most, but the B helix is similar to that of P450eryF, which leads to an enlarged active site when compared with P450 BM-3, P450cam, and P450terp. The 3A4 residues equivalent to known substrate contact residues of the bacterial proteins and key residues of rat P450 2B1 are located in the active site or the substrate access channel. Docking of progesterone into the P450 3A4 model demonstrated that the substrate bound in a 6-orientation can interact with a number of active site residues, such as 114, 119, 301, 304, 305, 309, 370, 373, and 479, through hydrophobic interactions. The active site of the enzyme can also accommodate erythromycin, which, in addition to the residues listed for progesterone, also contacts residues 101, 104, 105, 214, 215, 217, 218, 374, and 478. The majority of 3A4 residues which interact with progesterone and/or erythromycin possess their equivalents in key residues of P450 2B enzymes, except for residues 297, 480 and 482, which do not contact either substrate in P450 3A4. The results from docking of progesterone and erythromycin into the enzyme model make it possible to pinpoint residues which may be important for 3A4 function and to target them for site-directed mutagenesis.