Structure-Based Site-Directed Mutagenesis of Hydroxynitrile Lyase from Cyanogenic Millipede, Oxidus gracilis for Hydrocyanation and Henry Reactions.

Hydroxynitrile lyase (HNL) from the cyanogenic millipede Oxidus gracillis (OgraHNL) is a crucial enzyme in the cyanogenesis pathway. Here, the crystal structures of OgraHNL complexed with sulfate, benzaldehyde (BA), (R)-mandelonitrile ((R)-Man), (R)-2-chloromandelonitrile ((R)-2-Cl-Man), and acetone cyanohydrin (ACN) were solved at 1.6, 1.7, 2.3, 2.1, and 2.0 Šresolutions, respectively. The structure of OgraHNL revealed that it belonged to the lipocalin superfamily. Based on this structure, positive variants were designed to further improve the catalytic activity and enantioselectivity of the enzyme for asymmetric hydrocyanation and Henry reactions.

Kinetic Characterization of Estradiol Glucuronidation by Liver Microsomes and Expressed UGT Enzymes: The Effects of Organic Solvents.

BACKGROUND AND OBJECTIVE: In vitro glucuronidation of 17ß-estradiol (estradiol) is often performed to assess the role of uridine 5'-diphospho-glucuronosyltransferase 1A1 (UGT1A1) in xenobiotic/drug metabolism. The objective of this study was to determine the effects of four commonly used organic solvents [i.e., dimethyl sulfoxide (DMSO), methanol, ethanol, and acetonitrile] on the glucuronidation kinetics of estradiol, which can be glucuronidated at C3 and C17 positions. METHODS: The impacts of organic solvents on estradiol glucuronidation were determined by using expressed UGT enzymes and liver microsomes from both human and animals. RESULTS: In human liver microsomes (HLM), methanol, ethanol, and acetonitrile significantly altered estradiol glucuronidation kinetics with increased Vmax (up to 2.6-fold) and CLmax (up to 2.8-fold) values. Altered estradiol glucuronidation in HLM was deduced to be attributed to the enhanced metabolic activities of UGT1A1 and UGT2B7, whose activities differ at the two glucuronidation positions. The effects of organic solvents on estradiol glucuronidation were glucuronidation position-, isozyme-, and solvent-specific. Furthermore, both ethanol and acetonitrile have a greater tendency to modify the glucuronidation activity of estradiol in animal liver microsomes. CONCLUSION: Organic solvents such as methanol, ethanol, and acetonitrile showed great potential in adjusting the glucuronidation of estradiol. DMSO is the most suitable solvent due to its minimal influence on estradiol glucuronidation. Researchers should be cautious in selecting appropriate solvents to get accurate results when assessing the metabolism of a new chemical entity.

Molecular Composition and Biological Activity of a Novel Acetonitrile-Water Extract of Lens Culinaris Medik in Murine Native Cells and Cell Lines Exposed to Different Chemotherapeutics Using Mass Spectrometry.

We evaluated the effects of a new extract (70% acetonitrile, 2E0217022196DIPFARMTDA) of Lens culinaris Medik (Terre di Altamura SRL, Altamura BA) to prevent cytotoxic damage from cisplatin, staurosporine, irinotecan, doxorubicin, and the glucocorticoid dexamethasone. The acetonitrile-water extract (range 0.1-5 mg/mL) was obtained by extracting 10 g of lentil flour with 50 milliliters of the acetonitrile-water extraction mixture in a 70:30 ratio, first for 3 h and then overnight in a shaker at room temperature. The next day, the extract was filtered and passed through a Rotavapor to obtain only the aqueous component and eliminate that with acetonitrile, and then freeze-dried to finally have the powdered extract. In vitro experiments showed that the extract prevented the cytotoxic damage induced by cisplatin, irinotecan, and doxorubicin on HEK293 and SHSY5Y cell lines after 24-96 h. In murine osteoblasts after 24-72 h of incubation time, the extract was cytoprotective against all chemicals. The extract was effective against dexamethasone, leading to synergic cell proliferation in all cell types. In bone marrow cells, the extract is cytoprotective after 72 h against doxorubicin, staurosporine, and dexamethasone. Instead, on muscle fibers, the extract has a synergic effect with chemotherapeutics, increasing cytotoxicity induced by doxorubicin and staurosporine. LC-MS attested to the existence of several phenolic structures in the extract. The most abundant families of compounds were flavonoids (25.7%) and mellitic acid (18%). Thus, the development of this extract could be implemented in the area of research related to the chemoprevention of damage to renal, neuronal, bone marrow cells, and osteoblasts by chemotherapeutics; moreover, it could be used as a reinforcer of cytotoxic action of chemotherapeutics on muscle fibers.

Solving an MHC allele-specific bias in the reported immunopeptidome.

Identification of MHC class I-bound peptides by immunopurification of MHC complexes and subsequent analysis by mass spectrometry is crucial for understanding T cell immunology and immunotherapy. Investigation of the steps for the MHC ligand isolation process revealed biases in widely used isolation techniques toward peptides of lower hydrophobicity. As MHC ligand hydrophobicity correlates positively with immunogenicity, identification of more hydrophobic MHC ligands could potentially lead to more effective isolation of immunogenic peptides as targets for immunotherapies. We solved this problem by use of higher concentrations of acetonitrile for the separation of MHC ligands and their respective complexes. This increased overall MHC ligand identifications by 2-fold, increased detection of cancer germline antigen-derived peptides by 50%, and resulted in profound variations in isolation efficacy between different MHC alleles correlating with the hydrophobicity of their anchor residues. Overall, these insights enabled a more complete view of the immunopeptidome and overcame a systematic underrepresentation of these critical MHC ligands of high hydrophobicity.

Acetonitrile wastewater treatment enhanced by a hybrid membrane-aerated bioreactor containing aerated and non-aerated zones.

Acetonitrile (ACN) is a very volatile, toxic and nitrogen-rich organic compound. To enhance ACN wastewater treatment, a novel hybrid membrane-aerated bioreactor (MAB) containing aerated and non-aerated zones was established. A polypropylene hollow fiber membrane module (HF) and a silicone rubber membrane module (SR) were separately used as the bubble-free aeration diffuser and the biofilm carrier, and the non-aerated zones of these two types of reactors were packed with ceramsite. When the influent ACN loading was 1.200 kg/m3·d, under aeration pressures of 20 kPa in the HF-MAB and 40 kPa in the SR-MAB, ACN removal loadings of 1.116 kg/m3·d and 1.004 kg/m3·d, respectively, were achieved, and the TN (total nitrogen) removal loadings were 0.267 kg/m3·d and 0.246 kg/m3·d, respectively. In the MABs, different stratified biofilm structures of the two zones and the diffusion and counter-diffusion of oxygen synergistically promoted ACN degradation, nitrification and denitrification.

Development and validation of a sensitive LC-MS/MS method for determination of gefitinib and its major metabolites in human plasma and its application in non-small cell lung cancer patients.

Gefitinib, the first approved oral epidermal growth factor receptor (EGFR) inhibitor, has been demonstrated effective in cancers with EGFR active mutations. In this study, we established and validated a method for determining gefitinib and its main metabolites, M605211, M387783, M537194 and M523595 in patients with non-small cell lung cancer (NSCLC) by liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. The mobile phase was water: acetonitrile (35:65, v/v) with 0.1% formic acid at a flow-rate of 0.35 mL/min, within a 3 min run time. Gefitinib and its main metabolites were separated on a X-Terra RP18 column (50 × 2.1 mm, 3.5 µm) at 40 ℃ and subjected to mass analysis using positive electro-spray ionization (ESI). The calibration ranges of gefitinib and M523595 were 0.5-1000 ng/mL, and other compounds were 0.05-100 ng/mL with the correlation coefficients (r2) ≥ 0.99. Accuracies ranged from 92.60%-107.58 and the inter- and intra-assay precision were less than 15% for all analytes in quality control samples. There was no significant matrix effect. The ranges of extraction recoveries were 86-105% for all analytes and IS. Thirty plasmas were obtained from Sun Yat-sen university cancer center. The mean plasma concentration of (± SD) of gefitinib M537194, M523595, M387783 and M605211 were 247.18 (± 140.39) ng/mL, 7.78 (± 6.74) ng/mL, 101.09 (± 93.44) ng/mL, 1.6 (± 0.9) ng/mL and 11.63 (± 4.98) ng/mL, respectively. The validated LC/MS/MS method was effectively used in the determination of gefitinib and its four metabolites in NSCLC patients.

Prunasin production using engineered Escherichia coli expressing UGT85A47 from Japanese apricot and UDP-glucose biosynthetic enzyme genes.

Japanese apricot, Prunus mume Sieb. et Zucc., biosynthesizes the l-phenylalanine-derived cyanogenic glucosides prunasin and amygdalin. Prunasin has biological properties such as anti-inflammation, but plant extraction and chemical synthesis are impractical. In this study, we identified and characterized UGT85A47 from Japanese apricot. Further, UGT85A47 was utilized for prunasin microbial production. Full-length cDNA encoding UGT85A47 was isolated from Japanese apricot after 5'- and 3'-RACE. Recombinant UGT85A47 stoichiometrically catalyzed UDP-glucose consumption and synthesis of prunasin and UDP from mandelonitrile. Escherichia coli C41(DE3) cells expressing UGT85A47 produced prunasin (0.64 g/L) from racemic mandelonitrile and glucose. In addition, co-expression of genes encoding UDP-glucose biosynthetic enzymes (phosphoglucomutase and UTP-glucose 1-phosphate uridiltransferase) and polyphosphate kinase clearly improved prunasin production up to 2.3 g/L. These results showed that our whole-cell biocatalytic system is significantly more efficient than the existing prunasin production systems, such as chemical synthesis.

Development and validation of a liquid chromatography tandem mass spectrometry assay for AZD3965 in mouse plasma and tumor tissue: Application to pharmacokinetic and breast tumor xenograft studies.

AZD3965, a pyrole pyrimidine derivative, is a potent and orally bioavailable inhibitor of monocarboxylate transporter 1 (MCT1), currently in a Phase I clinical trial in UK for lymphomas and solid tumors. There is currently no published assay for AZD3965. The objectives of this study were to develop and validate a LC/MS/MS assay for quantifying AZD3965 in mouse plasma and tumor tissue. Protein precipitation with 0.1% formic acid in acetonitrile was used for sample preparation. Chromatographic separation was achieved on a C18 column followed by tandem mass spectrometry detection in multiple reaction monitoring mode with utilizing Atmospheric Pressure Chemical Ionization. AR-C155858 was used as the internal standard. The inter-day and intra-day precision and accuracy of quality control samples evaluated in plasma and tumor tissue were less than ±7% of the nominal concentrations. The extraction recovery, matrix effect and stability values were all within acceptable levels. Sample dilution integrity, accessed by diluting plasma spiked with AZD3965 10-fold with blank plasma, was 101%. The lower limit of quantification (LLOQ) and upper limit of quantification (ULOQ) were 0.15 ng/mL and 12 µg/mL, respectively, in plasma. The assay of AZD3965 in tumor tissue was also validated with good precision and accuracy. The LLOQ was 0.15 ng/mL in tumor tissue. This assay was successfully applied to pharmacokinetic and murine 4T1 breast tumor xenograft studies of AZD3965 in mice.

Analysing Cytochrome c Aggregation and Fibrillation upon Interaction with Acetonitrile: an in Vitro Study.

The propensity of native state to form aggregated and fibrillar assemblies is a hallmark of amyloidosis. Our study was focused at analyzing the aggregation and fibrillation tendency of cytochrome c in presence of an organic solvent i.e. acetonitrile. In vitro analysis revealed that the interaction of cytochrome c with acetonitrile facilitated the oligomerization of cytochrome c via the passage through an intermediate state which was obtained at 20 % v/v concentration of acetonitrile featured by a sharp hike in the ANS fluorescence intensity with a blue shift of 20 nm compared to the native state. Oligomers and fibrils were formed at 40 and 50 % v/v concentration respectively as indicated by a significant hike in the ThT fluorescence intensity, red shift of 55 nm in congo red binding assay and an increase in absorbance at 350 nm. They possess ß-sheet structure as evident from appearance of peak at 217 nm. Finally, authenticity of oligomeric and fibrillar species was confirmed by TEM imaging which revealed bead like aggregates and a meshwork of thread like fibrils respectively. It could be suggested that the fibrillation of bovine cytchrome c could serve as a model protein to unravel the general aggregation and fibrillation pattern of heme proteins. Graphical abstract ᅟ.

In vitro activation of dibromoacetonitrile to cyanide by myeloperoxidase.

Dibromoacetonitrile (DBAN) is a disinfection by-product classified as a potential human and animal carcinogen. This study aimed at investigating the ability of myeloperoxidase (MPO) to oxidize DBAN to cyanide (CN-) in vitro Detection of CN- served as a marker for the possible generation of free radical intermediates implicated in DBAN-induced toxicity. Optimum conditions for the oxidation of DBAN to CN- were characterized with respect to pH, temperature, and time of incubation as well as DBAN, MPO, potassium chloride, and hydrogen peroxide (H2O2) concentrations in incubation mixtures. Maximum reaction velocity and Michaelis-Menten constant were assessed. Addition of sodium hypochlorite to the reaction mixtures significantly enhanced the rate of the reaction. Addition of the MPO inhibitors, sodium azide, 4-amino benzoic acid hydrazine, or indomethacin to the reaction mixtures significantly decreased the rate of DBAN oxidation. Inclusion of the antioxidant enzyme superoxide dismutase in the incubation mixtures significantly decreased the rate of reaction. Inclusion of the sulfhydryl compounds as reduced glutathione, N-acetylcysteine, d-penicillamine, or l-cysteine enhanced the rate of DBAN oxidation. These results demonstrate the ability of MPO/H2O2/chloride ion system to oxidize DBAN to CN- and provide insight for the elucidation of DBAN chronic toxicity.

Exploitation of in silico potential in prediction, validation and elucidation of mechanism of anti-angiogenesis by novel compounds: comparative correlation between wet lab and in silico data.

This study describes in silico validation of the wet lab data from aeroplysinin-1, curcumin and halofuginone using Autodock Tools 4.0. The inhibition patterns of the vascular endothelial cell differentiation and capillary tube formation mediated by anti-angiogenic growth factors from these test compounds were found quite comparable with the in silico results using angiogenic targets (EFGR, bFGF and VEGFR-1). Successful validation of the wet lab results of the selected angiogenic targets by in silico method has led to exploit the hidden potential of in silico tools in preliminary screening of the unknown compounds for anti-angiogenic potential in a cost-effective manner.

Differential response to acrylonitrile toxicity in rat primary astrocytes and microglia.

Acrylonitrile (ACN) is a widely used chemical in the production of plastics, resins, nitriles, acrylic fibers, synthetic rubber and acrylamide. While acute high level exposures to ACN are known to be lethal, chronic low dose exposures causes glial cell tumors in rats. Recently, these glial tumors have been characterized as microglial in origin. While effects of ACN on astrocytes, the more numerous glial cell, have been investigated, the effects on microglia are unknown. This study was conducted to compare the responses of astrocytes and microglia to ACN treatment in vitro to address differential sensitivities and adaptive responses to this toxic chemical. Cell viability, ACN uptake, lipid peroxidation byproducts (F2-isoprostanes), glutathione (GSH) levels and expression of NF-E2-related factor 2 (Nrf2) were evaluated in primary rat microglia and astrocytes following ACN treatment. Results indicate that microglia are more sensitive to ACN than astrocytes, accumulating less ACN while demonstrating higher F2-isoprostane levels. GSH levels were up-regulated in both cell types, as a protective mechanism against ACN-induced oxidative stress, while Nrf2 levels were only induced in microglia. Our data suggest that microglia and astrocytes exhibit different sensitivities and responses to ACN, which are linked to the intracellular thiol status inherent to each of these cell types.

Determination of polycyclic aromatic hydrocarbons in edible seafood by QuEChERS-based extraction and gas chromatography-tandem mass spectrometry.

UNLABELLED: A high-throughput analysis of polycyclic aromatic hydrocarbons (PAHs) in edible seafood using QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) based extraction and gas chromatography-tandem mass spectrometry (GC-MS/MS) was developed and validated in 4 seafood matrices--crab, finfish, oyster, and shrimp. The extraction employs QuEChERS with dispersive solid phase extraction (dSPE) using cleanup sorbent that includes C18. The acetonitrile extract is partitioned into hexane and analyzed by GC-MS/MS. The procedure was validated by spike recovery experiments of 15 parent and 5 substituted PAHs at 5ppb and 25 ppb levels in the 4 matrices. Recoveries were 71%-130% with RSDs less than 14%. The limit of quantitation (LOQ) for benzo[a]pyrene are 0.4-2.5 ppb depending on matrix. For all 15 parent PAHs the LOQs are less than 10% of the Levels of Concern established by the U.S. Food and Drug Administration. Good recoveries were also achieved in aged spikes at 5 ppb level in oyster and shrimp. Method accuracy was further evaluated by analyzing NIST Standard Reference Material 1974b. Ten of 14 incurred PAHs corresponding to the parent compounds in the present study fell within the control limits established by the National Oceanic and Atmospheric Administration. Using the present method approximately 20 samples every 24 h can be analyzed with confirmation. PRACTICAL APPLICATION: The presented analytical method could be used for seafood safety assessments to examine petroleum contaminant levels in edible seafood after an oil spill. The method is fast and sensitive.

Toxicology and carcinogenesis studies of dibromoacetonitrile (CAS No. 3252-43-5) in F344/N rats and B6C3F1 mice (drinking water studies).

BACKGROUND: Dibromoacetonitrile occurs in chlorinated drinking water that contains bromine and interacts with organic matter.We studied the effects of dibromoacetonitrile in drinking water on male and female rats and mice to identify potential toxic or cancer-related hazards. METHODS: We gave drinking water containing 50, 100, or 200 milligrams of dibromoacetonitrile per liter of water to groups of 50 male and female rats and mice for two years. Control animals received the same tap water with no chemical added. At the end of the study, tissues from more than 40 sites were examined for every animal. RESULTS: Survival was similar for animals receiving dibromoacetonitrile and the controls. Male and female rats receiving dibromoacetonitrile had increased rates of squamous cell adenomas or carcinomas of the mouth (oral mucosa or tongue). Two male rats receiving 200 mg/L had rare adenomas of the glandular stomach. There were a few instances of skin tumors in female rats receiving 100 or 200 mg/L. Male and female mice exposed to dibromoacetonitrile had increased rates of squamous cell papillomas of the forestomach. There were also slight increases in the occurrence of liver tumors in male mice. CONCLUSIONS: We conclude that dibromoacetonitrile in the drinking water caused cancer of the oral cavity in male and female rats and of the glandular stomach in male rats. We conclude that dibromoacetonitrile caused cancer of the forestomach in male and female mice. Tumors of the skin in female rats and of the liver in male mice may also have been related to exposure to dibromoacetonitrile.

AnhE, a metallochaperone involved in the maturation of a cobalt-dependent nitrile hydratase.

Acetonitrile hydratase (ANHase) of Rhodococcus jostii RHA1 is a cobalt-containing enzyme with no significant sequence identity with characterized nitrile hydratases. The ANHase structural genes anhA and anhB are separated by anhE, predicted to encode an 11.1-kDa polypeptide. An anhE deletion mutant did not grow on acetonitrile but grew on acetamide, the ANHase reaction product. Growth on acetonitrile was restored by providing anhE in trans. AnhA could be used to assemble ANHase in vitro, provided the growth medium was supplemented with 50 microM CoCl(2). Ten- to 100-fold less CoCl(2) sufficed when anhE was co-expressed with anhA. Moreover, AnhA contained more cobalt when produced in cells containing AnhE. Chromatographic analyses revealed that AnhE existed as a monomer-dimer equilibrium (100 mm phosphate, pH 7.0, 25 degrees C). Divalent metal ions including Co(2+), Cu(2+), Zn(2+), and Ni(2+) stabilized the dimer. Isothermal titration calorimetry studies demonstrated that AnhE binds two half-equivalents of Co(2+) with K(d) of 0.12 +/- 0.06 nM and 110 +/- 35 nM, respectively. By contrast, AnhE bound only one half-equivalent of Zn(2+) (K(d) = 11 +/- 2 nM) and Ni(2+) (K(d) = 49 +/- 17 nM) and did not detectably bind Cu(2+). Substitution of the sole histidine residue did not affect Co(2+) binding. Holo-AnhE had a weak absorption band at 490 nM (epsilon = 9.7 +/- 0.1 m(-1) cm(-1)), consistent with hexacoordinate cobalt. The data support a model in which AnhE acts as a dimeric metallochaperone to deliver cobalt to ANHase. This study provides insight into the maturation of NHases and metallochaperone function.

Metabolism and disposition of [14C]dibromoacetonitrile in rats and mice following oral and intravenous administration.

1. Tissue distribution, metabolism, and disposition of oral (0.2-20 mg/kg) and intravenous (0.2 mg/kg) doses of [2-(14)C]dibromoacetonitrile (DBAN) were investigated in male rats and mice. 2. [(14)C]DBAN reacts rapidly with rat blood in vitro and binds covalently. Prior depletion of glutathione (GSH) markedly diminished loss of DBAN. Chemical reaction with GSH readily yielded glutathionylacetonitrile. 3. About 90% of the radioactivity from orally administered doses of [(14)C]DBAN was absorbed. After intravenous administration, 10% and 20% of the radioactivity was recovered in mouse and rat tissues, respectively, at 72 h. After oral dosing, three to four times less radioactivity was recovered, but radioactivity in stomach was mostly covalently bound. 4. Excretion of radioactivity into urine exceeded that in feces; 9-15% was exhaled as labeled carbon dioxide and 1-3% as volatiles in 72 h. 5. The major urinary metabolites were identified by liquid chromatography-mass spectrometry, and included acetonitrile mercaptoacetate (mouse), acetonitrile mercapturate, and cysteinylacetonitrile. 6.The primary mode of DBAN metabolism is via reaction with GSH, and covalent binding may be due to reaction with tissue sulphydryls.

Biocatalytic synthesis of (R)-(-)-mandelic acid from racemic mandelonitrile by cetyltrimethylammonium bromide-permeabilized cells of Alcaligenes faecalis ECU0401.

The nitrilase from Alcaligenes faecalis ECU0401 belongs to the category of arylacetonitrilase, which could hydrolyze 2-chloromandelonitrile, 3,4-dimethoxyphenylacetonitrile, mandelonitrile, and phenylacetonitrile into the corresponding arylacetic acids. To overcome the permeability barrier and prepare whole cell biocatalysts with high activities, permeabilization of Alcaligenes faecalis ECU0401 in relation to nitrilase activity was optimized by using cetyltrimethylammonium bromide (CTAB) as permeabilizing agent. The nitrilase activity from Alcaligenes faecalis ECU0401 increased 4.5-fold when the cells were permeabilized with 0.3% (w/v) CTAB for 20 min at 25 degrees C and pH 6.5. Consequently, almost all the mandelonitrile was consumed and converted to (R)-(-)-mandelic acid with greater than 99.9% enantiomeric excess (e.e.) by the CTAB-permeabilized cells. The permeability barrier has been significantly reduced in the hydrolysis of mandelonitrile by using CTAB-permeabilized cells and a dynamic resolution was successfully achieved, giving a 100% theoretical yield of (R)-(-)-mandelic acid. Efficient biocatalyst recycling was achieved as a result of cell immobilization in calcium alginate, with a product-to-biocatalyst ratio of 3.82 g (R)-(-)-mandelic acid g(-1) dry cell weight (dcw) cell after 20 cycles of repeated use.

Bioproduction of glycolic acid from glycolonitrile with a new bacterial isolate of Alcaligenes sp. ECU0401.

Alcaligenes sp. ECU0401 has been isolated from soil samples with high nitrilase activity against glycolonitrile using the enrichment culture technique. The preferred carbon/nitrogen sources and metal ions were sodium acetate, a composite of peptone and yeast extract, and Cu(2+), respectively. Glycolic acid was obtained in a yield of 96.5% after 14 h of biotransformation from a total of 200 mM glycolonitrile in the mode of sequential addition during the cultivation of Alcaligenes sp. ECU0401 in a 5-L jar fermenter. Fifty micromolars of glycolonitrile could be hydrolyzed in a yield of 94.1% by resting cells after 36 h. The microbial nitrilase system could hydrolyze various nitriles with high activity, and no amidase activity and glycolic acid were observed in hydrolyzing glycolamide. It significantly exhibited high enantioselectivity in the hydrolysis of mandelonitrile and 2-chloromandelonitrile (>99.9% e.e.( p )). Efficient biocatalyst recycling was achieved as a result of immobilization in glutaraldehyde/polyethylenimine cross-linked carrageenan with immobilized cells exhibiting a biocatalyst productivity of 1,042.2 g glycolic acid per gram dry cell weight after 29 batch recycles.

Isocyanides inhibit human heme oxygenases at the verdoheme stage.

Heme oxygenases (HO) catalyze the oxidative cleavage of heme to generate biliverdin, CO, and free iron. In humans, heme oxygenase-1 (hHO-1) is overexpressed in tumor tissues, where it helps to protect cancer cells from anticancer agents, while HOs in fungal pathogens, such as Candida albicans, function as the primary means of iron acquisition. Thus, HO can be considered a potential therapeutic target for certain diseases. In this study, we have examined the equilibrium binding of three isocyanides, isopropyl, n-butyl, and benzyl, to the two major human HO isoforms (hHO-1 and hHO-2), Candida albicans HO (CaHmx1), and human cytochrome P450 CYP3A4 using electronic absorption spectroscopy. Isocyanides coordinate to both ferric and ferrous HO-bound heme, with tighter binding by the more hydrophobic isocyanides and 200-300-fold tighter binding to the ferrous form. Benzyl isocyanide was the strongest ligand to ferrous heme in all the enzymes. Because the dissociation constants (KD) of the ligands for ferrous heme-hHO-1 were below the limit of accuracy for equilibrium titrations, stopped-flow kinetic experiments were used to measure the binding parameters of the isocyanides to ferrous hHO-1. Steady-state activity assays showed that benzyl isocyanide was the most potent uncompetitive inhibitor with respect to heme with a KI = 0.15 microM for hHO-1. Importantly, single turnover assays revealed that the reaction was completely stopped by coordination of the isocyanide to the verdoheme intermediate rather than to the ferric heme complex. Much tighter binding of the inhibitor to the verdoheme intermediate differentiates it from inhibition of, for example, CYP3A4 and offers a possible route to more selective inhibitor design.

Identification of amino acid residues responsible for the enantioselectivity and amide formation capacity of the Arylacetonitrilase from Pseudomonas fluorescens EBC191.

The nitrilase from Pseudomonas fluorescens EBC191 converted (R,S)-mandelonitrile with a low enantioselectivity to (R)-mandelic acid and (S)-mandeloamide in a ratio of about 4:1. In contrast, the same substrate was hydrolyzed by the homologous nitrilase from Alcaligenes faecalis ATCC 8750 almost exclusively to (R)-mandelic acid. A chimeric enzyme between both nitrilases was constructed, which represented in total 16 amino acid exchanges in the central part of the nitrilase from P. fluorescens EBC191. The chimeric enzyme clearly resembled the nitrilase from A. faecalis ATCC 8750 in its turnover characteristics for (R,S)-mandelonitrile and (R,S)-2-phenylpropionitrile (2-PPN) and demonstrated an even higher enantioselectivity for the formation of (R)-mandelic acid than the nitrilase from A. faecalis. An alanine residue (Ala165) in direct proximity to the catalytically active cysteine residue was replaced in the nitrilase from P. fluorescens by a tryptophan residue (as found in the nitrilase from A. faecalis ATCC 8750 and most other bacterial nitrilases) and several other amino acid residues. Those enzyme variants that possessed a larger substituent in position 165 (tryptophan, phenylalanine, tyrosine, or histidine) converted racemic mandelonitrile and 2-PPN to increased amounts of the R enantiomers of the corresponding acids. The enzyme variant Ala165His showed a significantly increased relative activity for mandelonitrile (compared to 2-PPN), and the opposite was found for the enzyme variants carrying aromatic residues in the relevant position. The mutant forms carrying an aromatic substituent in position 165 generally formed significantly reduced amounts of mandeloamide from mandelonitrile. The important effect of the corresponding amino acid residue on the reaction specificity and enantiospecificity of arylacetonitrilases was confirmed by the construction of a Trp164Ala variant of the nitrilase from A. faecalis ATCC 8750. This point mutation converted the highly R-specific nitrilase into an enzyme that converted (R,S)-mandelonitrile preferentially to (S)-mandeloamide.