Tailored poly(2-oxazoline) polymer brushes to control protein adsorption and cell adhesion

POx bottle-brush brushes (BBBs) are synthesized by SIPGP of 2-isopropenyl-2-oxazoline and consecutive LCROP of 2-oxazolines on 3-aminopropyltrimethoxysilane-modified silicon substrates. The side chain hydrophilicity and polarity are varied. The impact of the chemical composition and architecture of the BBB upon protein (fibronectin) adsorption and endothelial cell adhesion are investigated and prove extremely low protein adsorption and cell adhesion on BBBs with hydrophilic side chains such as poly(2-methyl-2-oxazoline) and poly(2-ethyl-2-oxazoline). The influence of the POx side chain terminal function upon adsorption and adhesion is minor but the side chain length has a significant effect on bioadsorption.     

Sensitivity and specificity of phenoloxidase reactions of 1059 strains and species of Micromycetes cultivated on malt/agar medium

The phenoloxidase (POx) activities of 1059 strains and species of micromycetes were determined on malt/agar medium. Overall, 600 (57%) of the isolates produced one or more POx. The sensitivity and specificity of the POx activities towards various substrates were used to group the isolates. Some 187 strains (31% of those producing POx) produced well-defined enzymes, 236 (39%) produced incompletely identified enzymes and 177 (30%) produced other, unidentified POx.     

Heterologous expression and biochemical characterization of novel pyranose 2-oxidases from the ascomycetes <Emphasis Type="Italic">Aspergillus nidulans</Emphasis> and <Emphasis Type="Italic">Aspergillus oryzae</Emphasis>

A gene encoding a pyranose 2-oxidase (POx; pyranose/oxygen 2-oxidoreductase; glucose 2-oxidase; EC 1.1.3.10) was identified in the genome of the ascomycete Aspergillus nidulans. Attempts to isolate POx directly from A. nidulans cultures or to homologously overexpress the native POx (under control of the constitutive gpdA promoter) in A. nidulans were unsuccessful. cDNA encoding POx was synthesized from mRNA and expressed in Escherichia coli, and the enzyme was subsequently purified and characterized. A putative pyranose 2-oxidase-encoding gene was also identified in the genome of Aspergillus oryzae. The coding sequence was synthetically produced and was also expressed in E. coli. Both purified enzymes were shown to be flavoproteins consisting of subunits of 65 kDa. The A. nidulans enzyme was biochemically similar to POx reported in literature. From all substrates, the highest catalytic efficiency was found with D-glucose. In addition, the enzyme catalyzes the two-electron reduction of 1,4-benzoquinone, several substituted benzoquinones and 2,6-dichloroindophenol. As judged by the catalytic efficiencies (k _cat/k _m), some of these quinone electron acceptors are better substrates for pyranose oxidase than oxygen. The enzyme from A. oryzae was physically similar but showed lower kinetic constants compared to the enzyme from A. nidulans. Distinct differences in the stability of the two enzymes may be attributed to a deletion and an insertion in the sequence, respectively.     

Characterization of pyranose oxidase variants for bioelectrocatalytic applications

Pyranose oxidase (POx) catalyzes the oxidation of d-glucose to 2-ketoglucose with concurrent reduction of oxygen to H₂O₂. POx from Trametes ochracea (ToPOx) is known to react with alternative electron acceptors including 1,4-benzoquinone (1,4-BQ), 2,6-dichlorophenol indophenol (DCPIP), and the ferrocenium ion. In this study, enzyme variants with improved electron acceptor turnover and reduced oxygen turnover were characterized as potential anode biocatalysts. Pre-steady-state kinetics of the oxidative half-reaction of ToPOx variants T166R, Q448H, L545C, and L547R with these alternative electron acceptors were evaluated using stopped-flow spectrophotometry. Higher kinetic constants were observed as compared to the wild-type ToPOx for some of the variants. Subsequently, the variants were immobilized on glassy carbon electrodes. Cyclic voltammetry measurements were performed to measure the electrochemical responses of these variants with glucose as substrate in the presence of 1,4-BQ, DCPIP, or ferrocene methanol as redox mediators. High catalytic efficiencies (I_max^app/K_M^app) compared to the wild-type POx proved the potential of these variants for future bioelectrocatalytic applications, in biosensors or biofuel cells. Among the variants, L545C showed the most desirable properties as determined kinetically and electrochemically.     

Oxidation of Phe454 in the Gating Segment Inactivates Trametes multicolor Pyranose Oxidase during Substrate Turnover

The flavin-dependent enzyme pyranose oxidase catalyses the oxidation of several pyranose sugars at position C-2. In a second reaction step, oxygen is reduced to hydrogen peroxide. POx is of interest for biocatalytic carbohydrate oxidations, yet it was found that the enzyme is rapidly inactivated under turnover conditions. We studied pyranose oxidase from Trametes multicolor (TmPOx) inactivated either during glucose oxidation or by exogenous hydrogen peroxide using mass spectrometry. MALDI-MS experiments of proteolytic fragments of inactivated TmPOx showed several peptides with a mass increase of 16 or 32 Da indicating oxidation of certain amino acids. Most of these fragments contain at least one methionine residue, which most likely is oxidised by hydrogen peroxide. One peptide fragment that did not contain any amino acid residue that is likely to be oxidised by hydrogen peroxide (DAFSYGAVQQSIDSR) was studied in detail by LC-ESI-MS/MS, which showed a +16 Da mass increase for Phe454. We propose that oxidation of Phe454, which is located at the flexible active-site loop of TmPOx, is the first and main step in the inactivation of TmPOx by hydrogen peroxide. Oxidation of methionine residues might then further contribute to the complete inactivation of the enzyme.     

Lead diversification. Application to existing drug molecules: mifepristone 1 and antalarmin 8

A series of C-H functionalisation plate-based chemical screens and other C-H activation protocols were developed for the chemical diversification of drug molecules. In this Letter, metalloporphyrin and other catalytic oxidation systems are described in addition to chlorination. Mifepristone and antalarmin are used as substrates. The products obtained and the biological data demonstrate the potential utility of this approach.     

New approaches for itaconic acid production: bottlenecks and possible remedies

Currently, growing attention is being devoted to the conversion of biomass into value-added products, such as itaconic acid (IA), which is considered as the cleanest alternative to petroleum-based acrylic acid. IA is an unsaturated dicarboxylic acid that is used as a building block chemical for the production of several value-added products such as poly-itaconic acid. IA and its derivatives have a wide range of potential applications in textile, paint, pharmaceutical and chemical industries. Presently, industries are producing IA on the large scale by fermentation from glucose. However, due to the primary utility of glucose as a food, it cannot meet the global demand for IA production in an economical way. The main challenge, so far, has been the production technology, which does not support cost-effective and competitive production of IA. This review discusses the various bottlenecks faced during each step of IA production, along with possible remedies to deal with these problems. Furthermore, it reviews the recent progress in fermentative IA production and sheds light on different microorganisms used, potential substrates and fermentation conditions. The review also covers market potential for IA, which indicates that IA can be produced cost-effectively from sustainable substrates, and it has the potential to replace petrochemicals in the near future.     

Alkane and wax ester production from lignin-related aromatic compounds

Lignin has potential as a sustainable feedstock for microbial production of industrially relevant molecules. However, the required lignin depolymerization yields a heterogenic mixture of aromatic monomers that are challenging substrates for the microorganisms commonly used in the industry. Here, we investigated the properties of lignin-related aromatic compounds (LRAs), namely coumarate, ferulate, and caffeate, in the synthesis of biomass and products in an LRA-utilizing bacterial host Acinetobacter baylyi ADP1. The biosynthesis products, wax esters, and alkanes are relevant compounds for the chemical and fuel industries. Here, wax esters were produced by a native pathway of ADP1, whereas alkanes were produced by a synthetic pathway introduced to the host. Using individual LRAs as substrates, the growth and product formation were monitored with internal biosensors and off-line analytics. Of the tested LRAs, coumarate was the most propitious in terms of product synthesis. Wax esters were produced from coumarate with yield and titer of 37 mg/g_coumarate and 202 mg/L, whereas alkanes were produced with a yield of 62.3 µg /g_coumarate and titer of 152 µg/L. This study demonstrates the microbial preference for certain LRAs and highlights the potential of A. baylyi ADP1 as a host for LRA upgrading to value-added products.     

Biocatalysis in natural products chemistry

The properties of enzymes and microbial cells as biocatalysts useful in natural products chemistry are discussed from the perspective of the chemical transformations they catalyse. Attention is focused on numerous reactions of value to natural products chemists, including the acyloin condensation, Baeyer-Villiger oxidation, regio- and enantioselective ester hydrolyses, oxidations of aromatic and non-aromatic substrates, oxidoreduction and O- and N-dealkylations. Compounds considered in this review include amino acids, alkaloids, antibiotics, coumarins, naphthoquinones, quassinoids, rotenoids and mono-, sesqui-, di- and triterpenoid substrates. The value of biocatalysis compared with traditional chemical catalysis is considered within the broad framework of natural products chemistry, and the potential for using immobilized enzyme and cell technology is presented.     

Enzyme catalyzed formation of radicals from S-adenosylmethionine and inhibition of enzyme activity by the cleavage products

A large superfamily of enzymes have been identified that make use of radical intermediates derived by reductive cleavage of S-adenosylmethionine. The primary nature of the radical intermediates makes them highly reactive and potent oxidants. They are used to initiate biotransformations by hydrogen atom abstraction, a process that allows a particularly diverse range of substrates to be functionalized, including substrates with relatively inert chemical structures. In the first part of this review, we discuss the evidence supporting the mechanism of radical formation from S-adenosylmethionine. In the second part of the review, we examine the potential of reaction products arising from S-adenosylmethionine to cause product inhibition. The effects of this product inhibition on kinetic studies of 'radical S-adenosylmethionine' enzymes are discussed and strategies to overcome these issues are reviewed. This article is part of a Special Issue entitled: Radical SAM enzymes and Radical Enzymology.     

Effects of Endogenous Substrates on Adaptation of Anaerobic Microbial Communities to 3-Chlorobenzoate

Lengthy adaptation periods in laboratory studies evaluating the potential for contaminant biodegradation in natural or engineered environments may indicate that the native microbial communities are not metabolizing the contaminants in situ. In this study, we characterized the adaptation period preceding the biodegradation of 3-chlorobenzoate in anaerobic communities derived from lake sediment and wastewater sludge digesters. The importance of alternative mechanisms of adaptation of the anaerobic communities to 3-chlorobenzoate was evaluated by monitoring the concentrations of metabolic substrates and products as well as the levels of total small subunit (SSU) rRNA and SSU rRNA from populations thought to be important in 3-chlorobenzoate mineralization. The anaerobic environments from which the 3-chlorobenzoate-degrading communities were derived contained different levels of endogenous substrates. Increasing methane levels in the digester and sediment communities and decreasing chemical oxygen demand concentrations in the sediment community during the adaptation periods revealed that endogenous substrates were preferentially utilized relative to 3-chlorobenzoate. Methane and chemical oxygen demand concentrations leveled off concomitantly with the onset of 3-chlorobenzoate biodegradation, suggesting that depletion of the preferentially degraded endogenous substrates stimulated 3-chlorobenzoate metabolism. Consistent with these observations, adaptation to 3-chlorobenzoate occurred more rapidly in digester samples that were depleted of endogenous substrates compared to samples that contained high levels of these biodegradable compounds. Other potential adaptation mechanisms, e.g., genetic change or selective population enrichment, appeared to be less important based on the reproducibility and relative lengths of the adaptation events, trends in the SSU rRNA levels, and/or amplification of SSU rRNA genes from key populations.     

Effects of endogenous substrates on adaptation of anaerobic microbial communities to 3-chlorobenzoate

Lengthy adaptation periods in laboratory studies evaluating the potential for contaminant biodegradation in natural or engineered environments may indicate that the native microbial communities are not metabolizing the contaminants in situ. In this study, we characterized the adaptation period preceding the biodegradation of 3-chlorobenzoate in anaerobic communities derived from lake sediment and wastewater sludge digesters. The importance of alternative mechanisms of adaptation of the anaerobic communities to 3-chlorobenzoate was evaluated by monitoring the concentrations of metabolic substrates and products as well as the levels of total small subunit (SSU) rRNA and SSU rRNA from populations thought to be important in 3-chlorobenzoate mineralization. The anaerobic environments from which the 3-chlorobenzoate-degrading communities were derived contained different levels of endogenous substrates. Increasing methane levels in the digester and sediment communities and decreasing chemical oxygen demand concentrations in the sediment community during the adaptation periods revealed that endogenous substrates were preferentially utilized relative to 3-chlorobenzoate. Methane and chemical oxygen demand concentrations leveled off concomitantly with the onset of 3-chlorobenzoate biodegradation, suggesting that depletion of the preferentially degraded endogenous substrates stimulated 3-chlorobenzoate metabolism. Consistent with these observations, adaptation to 3-chlorobenzoate occurred more rapidly in digester samples that were depleted of endogenous substrates compared to samples that contained high levels of these biodegradable compounds. Other potential adaptation mechanisms, e.g., genetic change or selective population enrichment, appeared to be less important based on the reproducibility and relative lengths of the adaptation events, trends in the SSU rRNA levels, and/or amplification of SSU rRNA genes from key populations.     

Free energy differences between enzyme bound states

A theory is presented that describes the free energy difference between the enzyme-substrate (ES) and enzyme-product (EP) complexes that is expected in enzymes optimized for catalytic efficiency. In such enzymes, the free energy drop between ES and EP complexes reflects a portion of the chemical potential difference between substrates and products outside the active site under physiological conditions. Qualitative and quantitative predictions of the model are discussed and compared with experimental data. The controversy over the kinetically optimal free energy profile for an enzymatic reaction operating under constraints set forward by Albery & Knowles (1976) is resolved.     

Molecular effects of nitrosamine toxicity.

Nitrosamines are toxic chemical compounds found low in quantity, but widespread in the environment. This work investigated the kinetics of chemical reaction of activated nitrosamines with various organic substrates. The mechanism by which nitrosamines react demonstrates possible pathways in which the toxicity is expressed. Once activated nitrosamines are very reactive. Chemical compounds which can act as nucleophilic substrates may be alkylated by the activated nitrosamines. A broad category of chemical compounds are shown to be suitable substrates for nitrosamine induced alkylation. This large category of substrates suggests a substantial potential for toxic activity in vivo. By investigating the reaction kinetics of activated nitrosamines a greater understanding of their toxic effects may be possible.     

Weedy lignocellulosic feedstock and microbial metabolic engineering: advancing the generation of ‘Biofuel’

Lignocellulosic materials are the most abundant renewable organic resources (~200 billion tons annually) on earth that are readily available for conversion to ethanol and other value-added products, but they have not yet been tapped for the commercial production of fuel ethanol. The lignocellulosic substrates include woody substrates such as hardwood (birch and aspen, etc.) and softwood (spruce and pine, etc.), agro residues (wheat straw, sugarcane bagasse, corn stover, etc.), dedicated energy crops (switch grass, and Miscanthus etc.), weedy materials (Eicchornia crassipes, Lantana camara etc.), and municipal solid waste (food and kitchen waste, etc.). Despite the success achieved in the laboratory, there are limitations to success with lignocellulosic substrates on a commercial scale. The future of lignocellulosics is expected to lie in improvements of plant biomass, metabolic engineering of ethanol, and cellulolytic enzyme-producing microorganisms, fullest exploitation of weed materials, and process integration of the individual steps involved in bioethanol production. Issues related to the chemical composition of various weedy raw substrates for bioethanol formation, including chemical composition-based structural hydrolysis of the substrate, need special attention. This area could be opened up further by exploring genetically modified metabolic engineering routes in weedy materials and in biocatalysts that would make the production of bioethanol more efficient.     

Chemical cytometry for monitoring metabolism of a Ras-mimicking substrate in single cells.

BACKGROUND: Chemical cytometry is an emerging technology that analyzes chemical contents of single cells by means of capillary electrophoresis or capillary chromatography. It has a potential to become an indispensable tool in analyses of heterogeneous cell populations such as those in tumors. Ras oncogenes are found in 30% of human cancers. To become fully functional products, oncogenic Ras proteins require at least three posttranslational modifications: farnesylation, endoproteolysis, and carboxyl-methylation. Therefore, enzymes that catalyze the three reactions, farnesyltransferase (FTase), endoprotease (EPase), and methyltransferase (MTase), are considered highly attractive therapeutic targets. In this work, we used chemical cytometry to study the metabolism of a pentapeptide substrate that can mimic Ras proteins with respect to their posttranslational modifications in solution. METHODS: Mouse mammary gland tumor cells (4T1) and mouse embryo fibroblasts (NIH3T3) were incubated with a fluorescently labeled pentapeptide substrate, 2',7'-difluorofluorescein-5-carboxyl-Gly-Cys-Val-Ilu-Ala. Cells were washed from the substrate and resuspended in phosphate buffered saline. Uptake of the substrate by the cells was monitored by laser scanning confocal microscopy. Single cells were injected into the capillary, lysed, and subjected to capillary electrophoresis. Fluorescent metabolic products were detected by laser-induced fluorescence and compared with products obtained by the conversion of the substrate by FTase, EPase, and MTase in solution. Co-sampling of single cells with the in-vitro products was used for such comparison. RESULTS: Confocal microscopy data showed that the substrate permeated the plasma membrane and clustered in the cytoplasm. Further capillary electrophoresis and chemical cytometry analyses showed that the substrate was converted into three fluorescently labeled products, two of which were secreted in the culture medium and one remained in the cells. The intracellular product was present at approximately 100,000 molecules per cell. The three metabolic products of the substrate were found to be different from the products of its processing by FTase, EPase, and MTase in solution. CONCLUSIONS: This is the first report of chemical cytometry in the context of Ras-signaling studies. The chemical cytometry method used in this work will find applications in the development of suitable peptide substrates for monitoring enzyme activities in single cells.     

Binding and Channeling of Alternative Substrates in the Enzyme DmpFG: a Molecular Dynamics Study

DmpFG is a bifunctional enzyme comprised of an aldolase subunit, DmpG, and a dehydrogenase subunit, DmpF. The aldehyde intermediate produced by the aldolase is channeled directly through a buried molecular channel in the protein structure from the aldolase to the dehydrogenase active site. In this study, we have investigated the binding of a series of progressively larger substrates to the aldolase, DmpG, using molecular dynamics. All substrates investigated are easily accommodated within the active site, binding with free energy values comparable to the physiological substrate 4-hydroxy-2-ketovalerate. Subsequently, umbrella sampling was utilized to obtain free energy surfaces for the aldehyde intermediates (which would be generated from the aldolase reaction on each of these substrates) to move through the channel to the dehydrogenase DmpF. Small substrates were channeled with limited barriers in an energetically feasible process. We show that the barriers preventing bulky intermediates such as benzaldehyde from moving through the wild-type protein can be removed by selective mutation of channel-lining residues, demonstrating the potential for tailoring this enzyme to allow its use for the synthesis of specific chemical products. Furthermore, positions of transient escape routes in this flexible channel were determined.     

Regioselectivity in the cytochromes P-450: control by protein constraints and by chemical reactivities

Three alicyclic compounds (D-camphor, adamantanone, adamantane) were found to be hydroxylated by the cytochrome P-450 isoenzymes P-450cam and P-450LM2. With P-450cam as the catalyst only one product was formed from each of the substrates: 5-exohydroxycamphor, 5-hydroxyadamantanone, and 1-adamantanol. With P-450LM2 as the catalyst, two or more isomeric products were formed from each substrate: 3-endo-, 5-exo-, and 5-endo-hydroxycamphor; 4-anti- and 5-hydroxyadamantanone; and 1- and 2- adamantanol. The products from P-450cam hydroxylations were found to be isosteric with one another, suggesting that each of them was attacked at a topologically congruent position within a rigid enzyme-substrate complex. The distribution of products from P-450LM2 hydroxylations, on the other hand, were similar to the distributions expected during solution-phase hydroxylations. Thus, it would appear that the complex which P-450LM2 forms with its substrate allows considerable movement of the substrate molecule, such that most of the hydrogens in the substrate are exposed to the enzymatic hydrogen abstractor. Under these conditions, the distribution of products more nearly reflects the rank order of chemical reactivities of the various hydroxylatable positions, with only a moderate protein-based steric constraint being expressed. These suggestions were also evident in the tightness of binding of the substrates to the two enzymes and in the magnitude of coupling between the substrate binding and the spin-state equilibria. Thus, the product from P-450cam-catalyzed hydroxylation may be predicted by a consideration of the relation of the topology of the prospective substrate to that of D-camphor. The products from P-450LM2-catalyzed hydroxylations, on the other hand, may be approximately predicted from the chemical reactivities of the various abstractable hydrogens in the prospective substrate.     

Enzyme catalyzed formation of radicals from S-adenosylmethionine and inhibition of enzyme activity by the cleavage products

A large superfamily of enzymes have been identified that make use of radical intermediates derived by reductive cleavage of S-adenosylmethionine. The primary nature of the radical intermediates makes them highly reactive and potent oxidants. They are used to initiate biotransformations by hydrogen atom abstraction, a process that allows a particularly diverse range of substrates to be functionalized, including substrates with relatively inert chemical structures. In the first part of this review, we discuss the evidence supporting the mechanism of radical formation from S-adenosylmethionine. In the second part of the review, we examine the potential of reaction products arising from S-adenosylmethionine to cause product inhibition. The effects of this product inhibition on kinetic studies of ‘radical S-adenosylmethionine’ enzymes are discussed and strategies to overcome these issues are reviewed. This article is part of a Special Issue entitled: Radical SAM enzymes and Radical Enzymology.     

Structure/redox potential relationship of simple organic compounds as potential precursors of dyes for laccase-mediated transformation

The aim of this study was to examine the ability of an extracellular fungal laccase (LAC) to form colored products from simple non-colored organic precursors. Thirty different phenolic and non-phenolic precursors (o-, m-, and p-methoxy-, hydroxy-, sulfonic-, and amino-substituted) were tested as single and coupled substrates in a LAC-catalyzed oxidation. The findings show that LAC catalyzes the formation of colored products (from yellow/brown to red and blue) by oxidation of single substrates that are benzene derivatives containing at least two substituents comprised of amino, hydroxy, and methoxy groups. All precursors were tested by cyclic voltammetry and the correlation between their structure and redox potential, and the possibility of their transformation into colored products by fungal LAC was found. Colored products were yielded from single substrates possessing a value of the oxidation peak (E(o)) lower than 1,150 mV vs. normal hydrogen electrode (NHE). Substrates with an oxidation peak higher than 1,150 mV vs. NHE were transformed by LAC into colored compounds only in the presence of an additional precursor characterized by a low value of E(o) and the presence of reactive substituents such as methoxy, hydroxy, and amino groups. Therefore, additional hydroxylation, methoxylation, and amination of phenolic and non-phenolic substrates may represent a strategy to increase the range of these compounds as potential dyes precursors.