ABSTRACT
In this study, we examined the Aureobasidium pullulans strains DSM 14940 and DSM 14941 included in the Blossom Protect™ agent to be used in the bioreduction reaction of a symmetrical dicarbonyl compound. Both chiral 2-hydroxy-1,2-diphenylethanone antipodes were obtained with a high enantiomeric purity. Mild conditions (phosphate buffer [pH 7.0, 7.2], 30 °C) were successfully employed in the synthesis of (S)-benzoin using two different methodologies: benzyl desymmetrization and rac-benzoin deracemization. Bioreduction carried out with higher reagent concentrations, lower pH values and prolonged reaction time, and in the presence of additives, enabled enrichment of the reaction mixture with (R)-benzoin. The described procedure is a potentially useful tool in the synthesis of chiral building blocks with a defined configuration in a simple and economical process with a lower environmental impact, enabling one-pot biotransformation.
Subject(s)
Aureobasidium/metabolism , Benzoin/metabolism , Benzoin/chemistry , Biocatalysis , Biotransformation , Hydrogen-Ion Concentration , Oxidation-Reduction , Phenylglyoxal/analogs & derivatives , Phenylglyoxal/chemistry , Phenylglyoxal/metabolism , StereoisomerismABSTRACT
Two novel types of non-aqueous bioconversion systems using fungal spores, either adsorbed on the surface of a filter pad or entrapped in calcium alginate beads, were constructed and applied for a model reaction: reduction of benzil to benzoin by Aspergillus sojae NBRC 32074. The spores adsorbed on a filter pad catalyzed the reduction in some toxic organic solvents, such as methylcyclohexane (log P: 3.61) and din-butyl ether (3.21). For the relationship between the reduction activity and the log P value of the organic solvent, a highly positive correlation (R2: 0.815) was observed. Surprisingly, the reduction proceeded in the more hydrophilic and toxic tert-butyl acetate (log P: 1.76). Glycerol was selected as the best hydride source. The higher the glycerol content, the more the benzoin was produced. While the production of benzil by spores was lower than that by mycelia in harmless di-n-hexyl ether (log P: 5.12), mycelia could not catalyze the reduction in the toxic tert-butyl acetate. In contrast, the spores entrapped in the calcium alginate beads could catalyze the reduction. Although the reduction by alginate-entrapped spores could be stably repeated 5 times in di-n-hexyl ether without a decline in the reduction activity, it was observed that the reduction activity of the spores gradually decreased after repeated reduction in tert-butyl acetate.
Subject(s)
Bioreactors , Spores, Fungal/metabolism , Acetates , Adsorption , Alginates , Aspergillus , Benzoin , Bioreactors/microbiology , Catalysis , Cyclohexanes , Ethers , Glucuronic Acid , Glycerol , Hexuronic Acids , Hydrophobic and Hydrophilic Interactions , Phenylglyoxal/analogs & derivatives , Phenylglyoxal/metabolism , SolventsABSTRACT
Modification with arginine-specific glyoxals modulates the permeability transition (PT) of rat liver mitochondria, with inhibitory or inducing effects that depend on the net charge of the adduct(s). Here, we show that phenylglyoxal (PGO) affects the PT in a species-specific manner (inhibition in mouse and yeast, induction in human and Drosophila mitochondria). Following the hypotheses (i) that the effects are mediated by conserved arginine(s) and (ii) that the PT is mediated by the F-ATP synthase, we have narrowed the search to 60 arginines. Most of these residues are located in subunits α, ß, γ, ϵ, a, and c and were excluded because PGO modification did not significantly affect enzyme catalysis. On the other hand, yeast mitochondria lacking subunit g or bearing a subunit g R107A mutation were totally resistant to PT inhibition by PGO. Thus, the effect of PGO on the PT is specifically mediated by Arg-107, the only subunit g arginine that has been conserved across species. These findings are evidence that the PT is mediated by F-ATP synthase.
Subject(s)
Arginine/metabolism , Mitochondria/metabolism , Mitochondrial Membrane Transport Proteins , Mitochondrial Proton-Translocating ATPases/metabolism , Phenylglyoxal/metabolism , Saccharomyces cerevisiae/metabolism , Animals , Calcium/metabolism , Catalysis , Dimerization , Drosophila , HEK293 Cells , Humans , Mice , Mitochondria/enzymology , Mitochondrial Permeability Transition Pore , Mitochondrial Proton-Translocating ATPases/chemistry , Species SpecificityABSTRACT
Human DHRS7 (SDR34C1) is one of insufficiently described enzymes of the short-chain dehydrogenase/reductase superfamily. The members of this superfamily often play an important pato/physiological role in the human body, participating in the metabolism of diverse substrates (e.g. retinoids, steroids, xenobiotics). A systematic approach to the identification of novel, physiological substrates of DHRS7 based on a combination of homology modeling, structure-based virtual screening and experimental evaluation has been used. Three novel substrates of DHRS7 (dihydrotestosterone, benzil and 4,4'-dimetylbenzil) have been described.
Subject(s)
Oxidoreductases/metabolism , Dihydrotestosterone/metabolism , Humans , Molecular Docking Simulation , Oxidoreductases/chemistry , Phenylglyoxal/analogs & derivatives , Phenylglyoxal/metabolism , Protein Binding , Protein ConformationABSTRACT
Plants are constantly exposed to numerous biotic or abiotic stress factors throughout their life-cycle. Pathogens and pathogen-derived molecules are the best studied inducers of plant defense responses, but synthetic and naturally occurring molecules have also been used to induce various types of resistance in plants. Here, an oxime molecule, 2-isonitrosoacetophenone (INAP), related to the stress metabolite citaldoxime, was used to trigger metabolic changes in the metabolome of treated Arabidopsis thaliana plants as monitored by UHPLC-MS in conjunction with principal component analysis (PCA) and orthogonal projection to latent structures discriminant analysis (OPLS-DA). The chemometric methods revealed metabolites found to be significantly present in response to the treatment. These include bioconversion products (2-keto-2-phenylacetaldoxime-glycoside and l-mandelonitrile-glycoside) as well as those of which the levels are affected by the treatment (benzoic acid and derivatives, other phenylpropanoid-derived compounds and glucosinolates). Using in planta bacterial growth evaluations, INAP treatment was furthermore found to induce an anti-microbial environment in vivo.
Subject(s)
Arabidopsis/drug effects , Arabidopsis/metabolism , Phenylglyoxal/analogs & derivatives , Benzoic Acid/pharmacology , Gene Expression Regulation, Plant/drug effects , Phenylglyoxal/metabolism , Phenylglyoxal/pharmacology , Principal Component AnalysisABSTRACT
The enzymatically active monomeric form of CuZn-superoxide dismutase has always been of interest to decipher the structure-function relationship in this class of enzymes. In the present study, spectroscopic and enzymatic characteristics of the dimeric and monomeric forms of recombinant Ipomoea carnea CuZn-superoxide dismutase were made to decipher their stability and altered catalytic properties. The monomeric form of protein was produced through site directed mutagenesis by replacing a conserved hydrophobic leucine with a polar lysine residue at the dimer-interface. Spectral characteristics of both the forms (monomer and dimer) showed the presence of novel electronic transitions. Superoxide scavenging activity of the mutated form was reduced to nearly half of the activity found in the native enzyme. Concomitantly, compared to native form the mutated enzyme showed an increase in peroxidase activity. High temperature dependent circular dichroism spectral analysis, differential scanning calorimetric profile, and the measurement of temperature dependent superoxide scavenging activity indicated an increased susceptibility of the mutated form to higher temperature as compared to the native form. The inhibitor studies like hydrogen peroxide, diethyldithiocarbamate and phenylglyoxal also indicate higher susceptibility, which might be due to, altered arrangement of active site residues as a consequence of the mutation. Molecular modeling and MD simulation studies further indicated that this specific mutation induces loss of hydrophobic interaction at dimer interface, resulting in the observed instability of the dimeric form. Increased peroxidative activity of the enzyme, upon monomerization may have physiological implication essentially in presence of high concentration of H2O2, as in case of plant cells specifically under stress conditions.
Subject(s)
Ipomoea/chemistry , Peroxidase/chemistry , Plant Proteins/chemistry , Superoxide Dismutase/chemistry , Catalytic Domain , Ditiocarb/chemistry , Ditiocarb/metabolism , Escherichia coli/enzymology , Escherichia coli/genetics , Gene Expression , Hydrogen Peroxide/chemistry , Hydrogen Peroxide/metabolism , Ipomoea/enzymology , Molecular Dynamics Simulation , Mutagenesis, Site-Directed , Oxidation-Reduction , Peroxidase/genetics , Peroxidase/metabolism , Phenylglyoxal/chemistry , Phenylglyoxal/metabolism , Plant Proteins/genetics , Plant Proteins/metabolism , Protein Multimerization , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Superoxide Dismutase/genetics , Superoxide Dismutase/metabolismABSTRACT
Glyoxalase 1 (GlxI) is the key enzyme that converts the highly reactive α-oxo-aldehydes into the corresponding α-hydroxy acids using l-glutathione as a cofactor. In our preliminary data, GlxI was identified as a substrate of transglutaminase 2 (TG2), a ubiquitous enzyme with multiple functions. According to the catalytic properties of TG2, protein cross-linking, polyamine conjugation, and/or deamidation are potential post-translational modifications. In this article, we have demonstrated that TG2 catalyzes either polyamine conjugation or deamidation to GlxI depending on the presence of polyamines or not. Deamidation leads to activation of GlxI while polyamine conjugation results in activation of GlxI as well as stabilization of GlxI against denaturation treatment. In cultured HeLa cells, methylglyoxal challenge causes increase in intracellular levels of reactive oxygen species (ROS) and calcium leading to TG2 activation and subsequent transamidation and activation of GlxI. The inhibition of TG2 significantly weakens the cell resistance to the methylglyoxal challenge. Thus, GlxI is a novel substrate of TG2 and is activated by TG2 in vitro and in cellulo. Exposure to methylglyoxal elicits a negative feedback loop entailing ROS, calcium, TG2 and GlxI, thus leading to attenuation of the increase in the methylglyoxal level. The results imply that cancer cells highly express TG2 or GlxI can endure the oxidative stress derived from higher glycolytic flux and may gain extra growth advantage from the aerobic glycolysis.
Subject(s)
GTP-Binding Proteins/metabolism , Lactoylglutathione Lyase/metabolism , Phenylglyoxal/metabolism , Transglutaminases/metabolism , Calcium/metabolism , Feedback, Physiological/drug effects , HeLa Cells , Humans , Polyamines/metabolism , Protein Glutamine gamma Glutamyltransferase 2 , Protein Processing, Post-Translational , Protein Stability , Reactive Oxygen Species/metabolismABSTRACT
Gene switches have wide utility in synthetic biology, gene therapy, and developmental biology, and multiple orthogonal gene switches are needed to construct advanced circuitry or to control complex phenotypes. Endogenous vascular endothelial growth factor (VEGF-A) is crucial to angiogenesis, and it has been shown that multiple alternately spliced VEGF-A isoforms are necessary for proper blood vessel formation. Such a necessity limits the utility of direct transgene delivery, which can provide only one splice variant. To overcome this limitation, we constructed a gene switch that can regulate the (VEGF-A) locus in mammalian cells by combining an engineered estrogen receptor (ER) ligand-binding domain (LBD), a p65 activation domain, and an artificial zinc-finger DNA binding domain (DBD). Our gene switch is specifically and reversibly controlled by 4,4'-dyhydroxybenzil (DHB), a small molecule, non-steroid synthetic ligand, which acts orthogonally in a mammalian system. After optimization of the gene switch architecture, an endogenous VEGF-A induction ratio of >100-fold can be achieved in HEK293 cells at 1 µM DHB, which is the highest endogenous induction reported to date. In addition, induction has been shown to be reversible, repeatable, and sustainable. Another advantage is that the ligand response is tunable by varying the clonal composition of a stably integrated cell line. The integration of our findings with the technology to change ligand specificity and DNA binding specificity will provide the framework for generating a wide array of orthogonal gene switches that can control multiple genes with multiple orthogonal ligands.
Subject(s)
Gene Expression Regulation , Genes, Switch , Synthetic Biology/methods , HEK293 Cells , Humans , Ligands , Neoplasm Proteins/genetics , Phenylglyoxal/analogs & derivatives , Phenylglyoxal/metabolism , Protein Structure, Tertiary , Receptors, Estrogen/genetics , Receptors, Estrogen/metabolism , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolismABSTRACT
The gene encoding a novel alcohol dehydrogenase that belongs to the short-chain dehydrogenases/reductases superfamily was identified in the aerobic thermoacidophilic crenarchaeon Sulfolobus acidocaldarius strain DSM 639. The saadh2 gene was heterologously overexpressed in Escherichia coli, and the resulting protein (SaADH2) was purified to homogeneity and both biochemically and structurally characterized. The crystal structure of the SaADH2 NADH-bound form reveals that the enzyme is a tetramer consisting of identical 27,024-Da subunits, each composed of 255 amino acids. The enzyme has remarkable thermophilicity and thermal stability, displaying activity at temperatures up to 80 °C and a 30-min half-inactivation temperature of â¼88 °C. It also shows good tolerance to common organic solvents and a strict requirement for NAD(H) as the coenzyme. SaADH2 displays a preference for the reduction of alicyclic, bicyclic and aromatic ketones and α-ketoesters, but is poorly active on aliphatic, cyclic and aromatic alcohols, showing no activity on aldehydes. Interestingly, the enzyme catalyses the asymmetric reduction of benzil to (R)-benzoin with both excellent conversion (98 %) and optical purity (98 %) by way of an efficient in situ NADH-recycling system involving a second thermophilic ADH. The crystal structure of the binary complex SaADH2-NADH, determined at 1.75 Å resolution, reveals details of the active site providing hints on the structural basis of the enzyme enantioselectivity.
Subject(s)
Archaeal Proteins/chemistry , Archaeal Proteins/metabolism , Fatty Acid Synthases/chemistry , Fatty Acid Synthases/metabolism , NADH, NADPH Oxidoreductases/chemistry , NADH, NADPH Oxidoreductases/metabolism , Phenylglyoxal/analogs & derivatives , Sulfolobus acidocaldarius/enzymology , Amino Acid Sequence , Archaeal Proteins/genetics , Benzoin/metabolism , Enzyme Stability , Fatty Acid Synthases/genetics , Hydrogen-Ion Concentration , Molecular Sequence Data , NADH, NADPH Oxidoreductases/genetics , Phenylglyoxal/metabolism , Sequence Homology, Amino Acid , Stereoisomerism , Substrate SpecificityABSTRACT
AIMS: Human NRDRB1, a 226 amino acid alternatively spliced isoform of the NADP(H)- dependent retinol dehydrogenase/reductase (NRDR), lacks the complete coding region of exon 3, but preserves all the important functional motifs for NRDR catalytic activity. Nevertheless, its tissue distribution and physiological function remain to be elucidated. METHODS: Expression of NRDRB1 and NRDR in cells and tissues was analyzed by semi-quantitative polymerase chain reaction (PCR) and western blot. NRDRB1 was expressed as a His(6) fusion protein and subjected to kinetics assays. RESULTS: Recombinant NRDRB1 had 1.2 to 8.6 fold higher k(cat)/K(m) values than recombinant NRDR, depending on the substrate. NRDRB1 catalyzed the NADPH-dependent reduction of α-dicarbonyl compounds, such as isatin, 9,10-phenanthrenequinone, and especially benzil. The significantly high catalytic activity and the relatively high expression in human liver of NRDRB1 conferred cellular resistance to benzil-induced cell toxicity and over-expression of NRDRB1 in low expressing Ec109 cells significantly enhanced cell tolerance toward benzil. CONCLUSIONS: Based on its substrate specificity, catalytic activity and relatively high expression in human liver tissue, our results suggest that NRDRB1, an alternatively spliced isoform of NRDR in vivo functions better than NRDR as a dicarbonyl reductase for xenobiotics containing reactive carbonyls. Our study is the first reporting this phenomenon of the enzymes involved in biochemical reactions.
Subject(s)
Oxidoreductases/metabolism , Phenylglyoxal/analogs & derivatives , Adult , Amino Acid Sequence , Cell Line, Tumor , Cell Survival/drug effects , Gene Expression , Humans , Inactivation, Metabolic , Isatin/chemistry , Isoenzymes/chemistry , Isoenzymes/genetics , Isoenzymes/metabolism , Kinetics , Liver/enzymology , Male , Middle Aged , Molecular Sequence Data , Organ Specificity , Oxidoreductases/chemistry , Oxidoreductases/genetics , Phenylglyoxal/chemistry , Phenylglyoxal/metabolism , Phenylglyoxal/pharmacology , Substrate SpecificityABSTRACT
Protein arginine deiminases (PADs) catalyze the hydrolysis of peptidyl arginine to form peptidyl citrulline. Abnormally high PAD activity is observed in a host of human diseases, but the exact role of protein citrullination in these diseases and the identities of specific citrullinated disease biomarkers remain unknown, largely because of the lack of readily available chemical probes to detect protein citrullination. For this reason, we developed a citrulline-specific chemical probe, rhodamine-phenylglyoxal (Rh-PG), which we show can be used to investigate protein citrullination. This methodology is superior to existing techniques because it possesses higher throughput and excellent sensitivity. Additionally, we demonstrate that this probe can be used to determine the kinetic parameters for a number of protein substrates, monitor drug efficacy, and identify disease biomarkers in an animal model of ulcerative colitis that displays aberrantly increased PAD activity.
Subject(s)
Citrulline/chemistry , Hydrolases/analysis , Molecular Probes/chemistry , Phenylglyoxal/chemistry , Animals , Biomarkers/analysis , Biomarkers/metabolism , Hydrolases/metabolism , Kinetics , Mice , Molecular Structure , Phenylglyoxal/metabolism , Rhodamines/blood , Rhodamines/chemistryABSTRACT
Nicotiana tabacum cell suspensions, 2 g wet wt/ml, rapidly took up 1 mM isonitrosoacetophenone (INAP), a plant-derived stress metabolite with anti-oxidative and anti-fungal properties, producing 4'-hexopyranosyloxy-3'-methoxyisonitrosoacetophenone in 54 % yield over 18 h. Unconverted INAP was at 33 µM. UPLC-MS/MS analyses with MassFragment software were used for metabolite identification. INAP had been hydroxylated at its meta- and para-positions as well as undergoing subsequent methoxylation and glycosylation. INAP is thus recognized by the enzymatic machinery of the phenylpropanoid pathway and is converted to a molecule with a substitution pattern similar to ferulic acid.
Subject(s)
Nicotiana/metabolism , Phenylglyoxal/analogs & derivatives , Biotransformation , Cells, Cultured , Chromatography, Liquid , Phenylglyoxal/metabolism , Tandem Mass SpectrometryABSTRACT
Phylogenetic analysis of 40 heme peroxidases, belonging to both prokaryotes and eukaryotes, revealed their clustering into three major classes. Class I represented sequences from plants, bacteria, fungi, and algae, whereas classes II and III exclusively represented plant and fungal peroxidases, respectively. Modeling of three representative classes of peroxidases, belonging to each of bacterial, plant, and fungal categories, revealed a similar kind of folding; however, superimposition analysis revealed relatively more closeness between plant and fungal peroxidases than that of the bacterial peroxidase. The docking analysis of three representative modeled peroxidases with three common substrates, namely, H2O2, guaiacol, and ascorbate, and three arginine-specific inhibitors, namely, phenylglyoxal, 1,2-cyclohexanedione, and 2,3-butanedione, revealed that all three inhibitors competed for guaiacol- and ascorbate-binding sites of peroxidases, except for phenylglyoxal binding in the case of plant peroxidase. Phenylglyoxal, 1,2-cyclohexanedione, and 2,3-butanedione were found to be most potent inhibitors of bacterial, fungal, and plant peroxidases, respectively.
Subject(s)
Catalytic Domain , Enzyme Inhibitors/pharmacology , Heme/metabolism , Molecular Docking Simulation , Peroxidases/antagonists & inhibitors , Peroxidases/chemistry , Phylogeny , Amino Acid Sequence , Bacteria/cytology , Bacteria/enzymology , Cell Membrane/metabolism , Cyclohexanones/metabolism , Cyclohexanones/pharmacology , Diacetyl/metabolism , Diacetyl/pharmacology , Enzyme Inhibitors/metabolism , Fungi/cytology , Fungi/enzymology , Molecular Sequence Data , Peroxidases/metabolism , Phenylglyoxal/metabolism , Phenylglyoxal/pharmacology , Plants/enzymology , Protein Transport , Solubility , Substrate SpecificityABSTRACT
Effective recognition and quantitative analysis of the prion protein are important in drug discovery and diagnosis for prion diseases, such as bovine spongiform encephalopathy and Creutzfeldt-Jakob diseases. We have developed a high-throughput method for a specific and sensitive determination of prion protein on a solid-phase membrane, based on a chemiluminescence reaction of aptamer with 3,4,5-trimethoxyphenylglyoxal. This method using aptamer is facile, inexpensive and convenient for the detection of the prion protein on a membrane, indicating a lower detection limit of ca. 4.2 pmol spot(-1).
Subject(s)
Aptamers, Nucleotide/metabolism , Biosensing Techniques/methods , Luminescent Measurements/methods , Membranes, Artificial , Phenylglyoxal/metabolism , Prions/analysis , Prions/chemistry , Animals , Aptamers, Nucleotide/genetics , Base Sequence , Cattle , High-Throughput Screening Assays , Immobilized Proteins/analysis , Immobilized Proteins/chemistry , MiceABSTRACT
The combination of chemical probing and high-resolution mass spectrometry constitutes a powerful alternative for the structural elucidation of biomolecules possessing unfavorable size, solubility, and flexibility. We have developed nested Arg-specific bifunctional crosslinkers to obtain complementary information to typical Cys- and Lys-specific reagents available on the market. The structures of 1,4-phenyl-diglyoxal (PDG) and 4,4'-biphenyl-diglyoxal (BDG) include two identical 1,2-dicarbonyl functions capable of reacting with the guanido group of Arg residues in proteins, as well as the base-pairing face of guanine in nucleic acids. The reactive functions are separated by modular spacers consisting of one or two benzene rings, which confer greater rigidity to the crosslinker structure than it is afforded by typical aliphatic spacers. Analysis by electrospray ionization (ESI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry has shown that the probes provide both mono- and bifunctional products with model protein substrates, which are stabilized by the formation of diester derivatives in the presence of borate buffer. The identification of crosslinked sites was accomplished by employing complementary proteolytic procedures and peptide mapping by ESI-FTICR. The results showed excellent correlation with the solvent accessibility and structural context of susceptible residues, and highlighted the significance of possible dynamic effects in determining the outcome of crosslinking reactions. The application of nested reagents with different spacing has provided a new tool for experimentally recognizing flexible regions that may be involved in prominent dynamics in solution. The development of new bifunctional crosslinkers with diverse target specificity and different bridging spans is expected to facilitate the structure elucidation of progressively larger biomolecular assemblies by increasing the number and diversity of spatial constraints available for triangulating the position of crosslinked structures in the three dimensions.
Subject(s)
Arginine/metabolism , Chemistry Techniques, Analytical/instrumentation , Cross-Linking Reagents/chemistry , Cross-Linking Reagents/metabolism , Proteins/chemistry , Proteins/metabolism , Amino Acid Sequence , Animals , Cattle , Humans , Models, Molecular , Phenylglyoxal/analogs & derivatives , Phenylglyoxal/chemistry , Phenylglyoxal/metabolism , Protein Conformation , Spectrometry, Mass, Electrospray Ionization , Substrate SpecificityABSTRACT
The asymmetric reduction of benzyl to (S)-benzoin with Penicillium claviforme IAM 7294 was applied to a liquid-liquid interface bioreactor (L-L IBR) using a unique polymeric material, ballooned microsphere (MS). The L-L IBR showed superior performance, as compared with suspension, organic-aqueous two-liquid-phase, and solid-liquid interface bioreactor (S-L IBR) systems, affording 14.4 g/l-organic phase of (S)-benzoin (99.0% ee).
Subject(s)
Benzoin/metabolism , Bioreactors/microbiology , Penicillium/metabolism , Phenylglyoxal/analogs & derivatives , Microspheres , Oxidation-Reduction , Penicillium/classification , Phenylglyoxal/metabolism , Water/chemistryABSTRACT
Human carboxylesterase 1 (hCE1) exhibits broad substrate specificity and is involved in xenobiotic processing and endobiotic metabolism. We present and analyze crystal structures of hCE1 in complexes with the cholesterol-lowering drug mevastatin, the breast cancer drug tamoxifen, the fatty acyl ethyl ester (FAEE) analogue ethyl acetate, and the novel hCE1 inhibitor benzil. We find that mevastatin does not appear to be a substrate for hCE1, and instead acts as a partially non-competitive inhibitor of the enzyme. Similarly, we show that tamoxifen is a low micromolar, partially non-competitive inhibitor of hCE1. Further, we describe the structural basis for the inhibition of hCE1 by the nanomolar-affinity dione benzil, which acts by forming both covalent and non-covalent complexes with the enzyme. Our results provide detailed insights into the catalytic and non-catalytic processing of small molecules by hCE1, and suggest that the efficacy of clinical drugs may be modulated by targeted hCE1 inhibitors.
Subject(s)
Anticholesteremic Agents/metabolism , Antineoplastic Agents, Hormonal/metabolism , Carboxylic Ester Hydrolases , Lovastatin/analogs & derivatives , Phenylglyoxal/analogs & derivatives , Protein Structure, Quaternary , Tamoxifen/metabolism , Acetates/chemistry , Acetates/metabolism , Anticholesteremic Agents/chemistry , Antineoplastic Agents, Hormonal/chemistry , Carboxylic Ester Hydrolases/antagonists & inhibitors , Carboxylic Ester Hydrolases/chemistry , Carboxylic Ester Hydrolases/metabolism , Crystallography, X-Ray , Humans , Lovastatin/chemistry , Lovastatin/metabolism , Models, Molecular , Molecular Sequence Data , Molecular Structure , Phenylglyoxal/chemistry , Phenylglyoxal/metabolism , Substrate Specificity , Tamoxifen/chemistryABSTRACT
Notexin, a presynaptic phospholipase A2 (PLA2) neurotoxin isolated from Notechis scutatus scutatus venom, was inactivated by arginine-specific reagents, phenylglyoxal and 1,2-cyclohexanedione. Kinetic analyses of the modification reaction revealed that the inactivation of notexin followed pseudo-first order kinetics and the loss of PLA2 activity was correlated with the incorporation of one molecule of modification reagent per toxin molecule. However, the results of amino acid analysis and sequence determination revealed that two arginine residues at positions 43 and 79 of notexin were modified simultaneously. Modification of the arginine residues was accompanied with a decrease in the ability to inhibit the indirectly evoked contraction of chick biventer cervicis muscle and bind with synaptic membranes. The secondary structure of the toxin molecule did not significantly change after modification with phenylglyoxal as revealed by the CD spectra. The modified derivative retained its affinity for Ca2+, indicating that the modified arginine residues did not participate in Ca2+ -binding. Together with the notion that Arg-43 and Arg-79 of notexin are located in the proximity of its catalytic site and toxic site, respectively, our results suggest that modification of Arg-43 and Arg-79 should differently contribute to the observed decrease in the PLA2 activity and neurotoxic effect of notexin.
Subject(s)
Arginine/metabolism , Elapid Venoms/metabolism , Phospholipases A/metabolism , Animals , Arginine/chemistry , Calcium/metabolism , Cervix Uteri/metabolism , Chickens , Circular Dichroism , Cyclohexanones/metabolism , Cyclohexanones/pharmacology , Elapid Venoms/chemistry , Elapid Venoms/toxicity , Female , Kinetics , Muscles/drug effects , Muscles/metabolism , Neurotoxins/chemistry , Neurotoxins/metabolism , Neurotoxins/toxicity , Phenylglyoxal/metabolism , Phenylglyoxal/pharmacology , Phospholipases A2 , Synaptic Membranes/drug effects , Synaptic Membranes/metabolismABSTRACT
Benzil (1) was selectively reduced to (S)-benzoin (2) in the presence of a wild-type Bacillus cereus Tim-r01. A 92% yield of 2 with 94% enantiomeric excess ratio was attained in phosphate-buffered saline (PBS) (pH 7.5) by using glucose as a nutrient at 37 degrees C for 12 h. Compound 2 was not reduced further to hydrobenzoin (3) at all. The reduction activity differed greatly depending on the strain of B. cereus. Under these conditions the B. cereus strains IFO3001, IFO15305, IAM1110, IAM1229, IAM1656, and IAM1729 gave 2 in yields ranging from 23 to 46% and the configuration of 2 was (S)-form (7 to 86% ee).
Subject(s)
Bacillus cereus/enzymology , Benzoin/chemistry , Phenylglyoxal/analogs & derivatives , Phenylglyoxal/chemistry , Bacillus cereus/genetics , Benzoin/metabolism , Buffers , Culture Media , Glucose/pharmacology , Hydrogen-Ion Concentration , Oxidation-Reduction , Phenylglyoxal/metabolism , Stereoisomerism , Temperature , Time FactorsABSTRACT
The chemical modifications of rabbit liver carbonyl reductase (RLCR) with phenylglyoxal (PGO) and 2,3,4-trinitrobenzenesulfonate sodium (TNBS), which are respective chemical modifiers of arginine and lysine residues, were examined. RLCR was rapidly inactivated by these modifiers. Kinetic data for the inactivation demonstrated that each one of arginine and lysine residues is essential for catalytic activity of the enzyme. Furthermore, based on the protective effects of NADP+, NAD+ and their constituents against the inactivation of RLCR by PGO and TNBS, we propose the possibility that the functional arginine and lysine residues are located in the coenzyme-binding domain of RLCR and interact with the 2'-phosphate group of NADPH.