Biocatalytic Synthesis of α-Amino Esters via Nitrene C-H Insertion.

α-Amino esters are precursors to noncanonical amino acids used in developing small-molecule therapeutics, biologics, and tools in chemical biology. α-C-H amination of abundant and inexpensive carboxylic acid esters through nitrene transfer presents a direct approach to α-amino esters. Methods for nitrene-mediated amination of the protic α-C-H bonds in carboxylic acid esters, however, are underdeveloped. This gap arises because hydrogen atom abstraction (HAA) of protic C-H bonds by electrophilic metal-nitrenoids is slow: metal-nitrenoids preferentially react with polarity-matched, hydridic C-H bonds, even when weaker protic C-H bonds are present. This study describes the discovery and evolution of highly stable protoglobin nitrene transferases that catalyze the enantioselective intermolecular amination of the α-C-H bonds in carboxylic acid esters. We developed a high-throughput assay to evaluate the activity and enantioselectivity of mutant enzymes together with their sequences using the Every Variant Sequencing (evSeq) method. The assay enabled the identification of enantiodivergent enzymes that function at ambient conditions in Escherichia coli whole cells and whose activities can be enhanced by directed evolution for the amination of a range of substrates.

Kinetic and structural analysis of redox-reversible artificial imine reductases.

Three artificial imine reductases, constructed via supramolecular anchoring utilising FeIII-azotochelin, a natural siderophore, to bind an iridium-containing catalyst to periplasmic siderophore-binding protein (PBP) scaffolds, have previously been synthesised and subjected to catalytic testing. Despite exhibiting high homology and possessing conserved siderophore anchor coordinating residues, the three artificial metalloenzymes (ArMs) displayed significant variability in turnover frequencies (TOFs). To further understand the catalytic properties of these ArMs, their kinetic behaviour was evaluated with respect to the reduction of three cyclic imines: dihydroisoquinoline, harmaline, and papaverine. Kinetic analyses revealed that all examined ArMs adhere to Michaelis-Menten kinetics, with the most pronounced saturation profile observed for the substrate harmaline. Additionally, molecular docking studies suggested varied hydrogen-bonding interactions between substrates and residues within the artificial binding pocket. Pi-stacking and pi-cation interactions were identified for harmaline and papaverine, corroborating the higher affinity of these substrates for the ArMs in comparison to dihydroisoquinoline. Furthermore, it was demonstrated that multiple cavities are capable of accommodating substrates in close proximity to the catalytic centre, thereby rationalising the moderate enantioselectivity conferred by the unmodified scaffolds.

Biocatalytic Synthesis of Ruxolitinib Intermediate via Engineered Imine Reductase.

An enzyme catalyzed strategy for the synthesis of a chiral hydrazine from 3-cyclopentyl-3-oxopropanenitrile 5 and hydrazine hydrate 2 is presented. An imine reductase (IRED) from Streptosporangium roseum was identified to catalyze the reaction between 3-cyclopentyl-3-oxopropanenitrile 5 and hydrazine hydrate 2 to produce trace amounts of (R)-3-cyclopentyl-3-hydrazineylpropanenitrile 4. We employed a 2-fold approach to optimize the catalytic performance of this enzyme. First, a transition state analogue (TSA) model was constructed to illuminate the enzyme-substrate interactions. Subsequently, the Enzyme_design and Funclib methods were utilized to predict mutants for experimental evaluation. Through three rounds of site-directed mutagenesis, site saturation mutagenesis, and combinatorial mutagenesis, we obtained mutant M6 with a yield of 98% and an enantiomeric excess (ee) of 99%. This study presents an effective method for constructing a hydrazine derivative via IRED-catalyzed reductive amination of ketone and hydrazine. Furthermore, it provides a general approach for constructing suitable enzymes, starting from nonreactive enzymes and gradually enhancing their catalytic activity through active site modifications.

Structure of the iminium reaction intermediate in an engineered aldolase explains the carboligation activity toward arylated ketones and aldehydes.

Two structures of fructose 6-phosphate aldolase, the wild-type and an engineered variant containing five active-site mutations, have been solved by cryoelectron microscopy (cryo-EM). The engineered variant affords production of aldols from aryl substituted ketones and aldehydes. This structure was solved to a resolution of 3.1 Å and contains the critical iminium reaction intermediate trapped in the active site. This provides new information that rationalizes the acquired substrate scope and aids in formulating hypotheses of the chemical mechanism. A Tyr residue (Y131) is positioned for a role as catalytic acid/base during the aldol reaction and the different structures demonstrate mobility of this amino acid residue. Further engineering of this fructose 6-phosphate aldolase (FSA) variant, guided by this new structure, identified additional FSA variants that display improved carboligation activities with 2-hydroxyacetophenone and phenylacetaldehyde.

Enzymatic Synthesis of Unprotected α,ß-Diamino Acids via Direct Asymmetric Mannich Reactions.

α,ß-Diamino acids are important structural motifs and building blocks for numerous bioactive natural products, peptidomimetics, and pharmaceuticals, yet efficient asymmetric synthesis to access these stereoarrays remains a challenge. Herein, we report the development of a pyridoxal 5'-phosphate (PLP)-dependent enzyme that is engineered to catalyze stereoselective Mannich-type reactions between free α-amino acids and enolizable cyclic imines. This biocatalyst enabled one-step asymmetric enzymatic synthesis of the unusual pyrrolidine-containing amino acid L-tambroline at gram-scale with high enantio- and diastereocontrol. Furthermore, this enzymatic platform is capable of utilizing a diverse range of α-amino acids as the Mannich donor and various cyclic imines as the acceptor. By coupling with different imine-generating enzymes, we established versatile biocatalytic cascades and demonstrated a general, concise, versatile, and atom-economic approach to access unprotected α,ß-diamino acids, including structurally complex α,α-disubstituted α,ß-diamino acids with contiguous stereocenters.

Atroposelective Synthesis of Axial Biaryls by Dynamic Kinetic Resolution Using Engineered Imine Reductases.

Axially chiral biaryl compounds are ubiquitous scaffolds in natural products, bioactive molecules, chiral ligands and catalysts, but biocatalytic methods for their asymmetric synthesis are limited. Herein, we report a highly efficient biocatalytic route for the atroposelective synthesis of biaryls by dynamic kinetic resolution (DKR). This DKR approach features a transient six-membered aza-acetal-bridge-promoted racemization followed by an imine reductase (IRED)-catalyzed stereoselective reduction to construct the axial chirality under ambient conditions. Directed evolution of an IRED from Streptomyces sp. GF3546 provided a variant (S-IRED-Ss-M11) capable of catalyzing the DKR process to access a variety of biaryl aminoalcohols in high yields and excellent enantioselectivities (up to 98 % yield and >99 : 1 enantiomeric ratio). Molecular dynamics simulation studies on the S-IRED-Ss-M11 variant revealed the origin of its improved activity and atroposelectivity. By exploiting the substrate promiscuity of IREDs and the power of directed evolution, our work further extends the biocatalysts' toolbox to construct challenging axially chiral molecules.

Iminium ion metabolites are formed from nintedanib by human CYP3A4.

Nintedanib is used to treat idiopathic pulmonary fibrosis, systemic sclerosis, interstitial lung disease, and progressive fibrotic interstitial lung disease. It is primarily cleared via hepatic metabolism, hydrolysis, and glucuronidation. In addition, formation of the iminium ion, a possible reactive metabolite, was predicted based on the chemical structure of nintedanib. To obtain a hint which may help to clarify the cause of nintedanib-induced liver injury, we investigated whether iminium ions were formed in the human liver. To detect unstable iminium ions using liquid chromatography-tandem mass spectrometry (LC-MS/MS), potassium cyanide was added to the reaction mixture as a trapping agent. Human liver and intestinal microsomes were incubated with nintedanib in the presence of NADPH to form two iminium ion metabolites on the piperazine ring. Their formation is strongly inhibited by ketoconazole, a potent cytochrome P450 (CYP) 3A4 inhibitor. Among the recombinant P450s, only CYP3A4 formed cyanide adducts. The role of CYP3A4 was supported by the positive correlation between CYP3A4 protein abundance, as determined by LC-MS-based proteomics, and the formation of cyanide adducts in 25 individual human liver microsomes. In conclusion, we have demonstrated that iminium ion metabolites are formed from nintedanib by CYP3A4 as potential reactive metabolites.

Designing Michaelases: exploration of novel protein scaffolds for iminium biocatalysis.

Biocatalysis is becoming a powerful and sustainable alternative for asymmetric catalysis. However, enzymes are often restricted to metabolic and less complex reactivities. This can be addressed by protein engineering, such as incorporating new-to-nature functional groups into proteins through the so-called expansion of the genetic code to produce artificial enzymes. Selecting a suitable protein scaffold is a challenging task that plays a key role in designing artificial enzymes. In this work, we explored different protein scaffolds for an abiological model of iminium-ion catalysis, Michael addition of nitromethane into E-cinnamaldehyde. We studied scaffolds looking for open hydrophobic pockets and enzymes with described binding sites for the targeted substrate. The proteins were expressed and variants harboring functional amine groups - lysine, p-aminophenylalanine, or N6-(D-prolyl)-L-lysine - were analyzed for the model reaction. Among the newly identified scaffolds, a thermophilic ene-reductase from Thermoanaerobacter pseudethanolicus was shown to be the most promising biomolecular scaffold for this reaction.

A triple cysteine motif as major determinant of the modulation of neuronal KV7 channels by the paracetamol metabolite N-acetyl-p-benzo quinone imine.

BACKGROUND AND PURPOSE: The analgesic action of paracetamol involves KV7 channels, and its metabolite N-acetyl-p-benzo quinone imine (NAPQI), a cysteine modifying reagent, was shown to increase currents through such channels in nociceptors. Modification of cysteine residues by N-ethylmaleimide, H2O2, or nitric oxide has been found to modulate currents through KV7 channels. The study aims to identify whether, and if so which, cysteine residues in neuronal KV7 channels might be responsible for the effects of NAPQI. EXPERIMENTAL APPROACH: To address this question, we used a combination of perforated patch-clamp recordings, site-directed mutagenesis, and mass spectrometry applied to recombinant KV7.1 to KV7.5 channels. KEY RESULTS: Currents through the cardiac subtype KV7.1 were reduced by NAPQI. Currents through all other subtypes were increased, either by an isolated shift of the channel voltage dependence to more negative values (KV7.3) or by such a shift combined with increased maximal current levels (KV7.2, KV7.4, KV7.5). A stretch of three cysteine residues in the S2-S3 linker region of KV7.2 was necessary and sufficient to mediate these effects. CONCLUSION AND IMPLICATION: The paracetamol metabolite N-acetyl-p-benzo quinone imine (NAPQI) modifies cysteine residues of KV7 subunits and reinforces channel gating in homomeric and heteromeric KV7.2 to KV7.5, but not in KV7.1 channels. In KV7.2, a triple cysteine motif located within the S2-S3 linker region mediates this reinforcement that can be expected to reduce the excitability of nociceptors and to mediate antinociceptive actions of paracetamol.

Cyanide Trapping of Iminium Ion Reactive Metabolites: Implications for Clinical Hepatotoxicity.

Reactive metabolite formation is a major mechanism of hepatotoxicity. Although reactive electrophiles can be soft or hard in nature, screening strategies have generally focused on the use of glutathione trapping assays to screen for soft electrophiles, with many data sets available to support their use. The use of a similar assay for hard electrophiles using cyanide as the trapping agent is far less common, and there is a lack of studies with sufficient supporting data. Using a set of 260 compounds with a defined hepatotoxicity status by the FDA, a comprehensive literature search yielded cyanide trapping data on an unbalanced set of 20 compounds that were all clinically hepatotoxic. Thus, a further set of 19 compounds was selected to generate cyanide trapping data, resulting in a more balanced data set of 39 compounds. Analysis of the data demonstrated that the cyanide trapping assay had high specificity (92%) and a positive predictive value (83%) such that hepatotoxic compounds would be confidently flagged. Structural analysis of the adducts formed revealed artifactual methylated cyanide adducts to also occur, highlighting the importance of full structural identification to confirm the nature of the adduct formed. The assay was demonstrated to add the most value for compounds containing typical structural alerts for hard electrophile formation: half of the severe hepatotoxins with these structural alerts formed cyanide adducts, while none of the severe hepatotoxins with no relevant structural alerts formed adducts. The assay conditions used included cytosolic enzymes (e.g., aldehyde oxidase) and an optimized cyanide concentration to minimize the inhibition of cytochrome P450 enzymes by cyanide. Based on the demonstrated added value of this assay, it is to be initiated for use at GSK as part of the integrated hepatotoxicity strategy, with its performance being reviewed periodically as more data is generated.

An imine reductase that captures reactive intermediates in the biosynthesis of the indolocarbazole reductasporine.

Imine reductases (IREDs) and reductive aminases have been used in the synthesis of chiral amine products for drug manufacturing; however, little is known about their biological contexts. Here we employ structural studies and site-directed mutagenesis to interrogate the mechanism of the IRED RedE from the biosynthetic pathway to the indolocarbazole natural product reductasporine. Cocrystal structures with the substrate-mimic arcyriaflavin A reveal an extended active site cleft capable of binding two indolocarbazole molecules. Site-directed mutagenesis of a conserved aspartate in the primary binding site reveals a new role for this residue in anchoring the substrate above the NADPH cofactor. Variants targeting the secondary binding site greatly reduce catalytic efficiency, while accumulating oxidized side-products. As indolocarbazole biosynthetic intermediates are susceptible to spontaneous oxidation, we propose the secondary site acts to protect against autooxidation, and the primary site drives catalysis through precise substrate orientation and desolvation effects. The structure of RedE with its extended active site can be the starting point as a new scaffold for engineering IREDs and reductive aminases to intercept large substrates relevant to industrial applications.

Evidence for cytochrome P450 3A4-mediated metabolic activation of SCO-267.

SCO-267 is a potent G-protein-coupled receptor 40 agonist that is undergoing clinical development for the treatment of type 2 diabetes mellitus. The current work was undertaken to investigate the bioactivation potential of SCO-267 in vitro and in vivo. Three SCO-267-derived glutathione (GSH) conjugates (M1-M3) were found both in rat and human liver microsomal incubations supplemented with GSH and nicotinamide adenine dinucleotide phosphate. Two GSH conjugates (M1-M2) together with two N-acetyl-cysteine conjugates (M4-M5) were detected in the bile of rats receiving SCO-267 at 10 mg/kg. The identified conjugates suggested the generation of quinone-imine and ortho-quinone intermediates. CYP3A4 was demonstrated to primarily catalyze the bioactivation of SCO-267. In addition, SCO-267 concentration-, time-, and NADPH-dependently inactivated CYP3A in human liver microsomes using testosterone as a probe substrate, along with KI and kinact values of 4.91 µM and 0.036 min-1 , respectively. Ketoconazole (a competitive inhibitor of CYP3A) displayed no significant protective effect on SCO-267-induced CYP3A inactivation. However, inclusion of GSH showed significant protection. These findings revealed that SCO-267 undergoes a facile CYP3A4-catalyzed bioactivation with the generation of quinone-imine and ortho-quinone intermediates, which were assumed to be involved in SCO-267 induced CYP3A inactivation. These findings provide further insight into the bioactivation pathways involved in the generation of reactive, potentially toxic metabolites of SCO-267. Further studies are needed to evaluate the influence of SCO-267 metabolism on the safety of this drug in vivo.

Asymmetric Synthesis of Sterically Hindered 1-Substituted Tetrahydro-ß-carbolines Enabled by Imine Reductase: Enzyme Discovery, Protein Engineering, and Reaction Development.

We report the discovery of a new imine reductase (IRED), named AtIRED, by genome mining. Site-saturation mutagenesis on AtIRED generated two single mutants M118'L and P120'G and the double mutant M118'L/P120'G with improved specific activity toward sterically hindered 1-substituted dihydro-ß-carbolines. The synthetic potential of these engineered IREDs was showcased by the preparative-scale synthesis of nine chiral 1-substituted tetrahydro-ß-carbolines (THßCs), including (S)-1-t-butyl-THßC and (S)-1-t-pentyl-THßC, in 30-87% isolated yields with excellent optical purities (98-99% ee).

Evidence for Metabolic Activation of Omeprazole In Vitro and In Vivo.

Omeprazole (OPZ) is a proton pump inhibitor commonly used for the treatment of gastric acid hypersecretion. Studies have revealed that use of OPZ can induce hepatotoxicity, but the mechanisms by which it induces liver injury are unclear. This study aimed to identify reactive metabolites of OPZ, determine the pathways of the metabolic activation, and define the correlation of the bioactivation with OPZ cytotoxicity. Quinone imine-derived glutathione (GSH), N-acetylcysteine (NAC), and cysteine (Cys) conjugates were detected in OPZ-fortified rat and human liver microsomal incubations captured with GSH, NAC, or Cys. The same GSH conjugates were detected in bile of rats and cultured liver primary cells after exposure to OPZ. Similarly, the same NAC conjugates were detected in urine of OPZ-treated rats. The resulting quinone imine was found to react with Cys residues of hepatic protein. CYP3A4 dominated the metabolic activation of OPZ. Exposure to OPZ resulted in decreased cell survival in cultured primary hepatocytes. Pretreatment with ketoconazole attenuated the susceptibility of hepatocytes to the cytotoxicity of OPZ.

miR-126-3p-loaded small extracellular vesicles secreted by urine-derived stem cells released from a phototriggered imine crosslink hydrogel could enhance vaginal epithelization after vaginoplasty.

BACKGROUND: Due to the large area and deep width of the artificial neovagina after vaginoplasty, it takes a considerable amount of time to achieve complete epithelization of the neovagina. Currently, the clinical therapies for vaginal epithelization after vaginoplasty are still dissatisfactory. Recent studies showed that small extracellular vesicles (sEVs) derived from stem cells could accelerate wound epithelization. The sustained release of sEVs from optimized hydrogels may be a promising strategy to accelerate vaginal epithelization after vaginoplasty. METHODS: The efficacy of phototriggered imine crosslink hydrogels (piGEL) containing sEVs derived from human urine-derived stem cells (hUSC-sEVs, piGEL-sEVs) on vaginal mucosa defects in rabbits was assessed by wound closure rates, histological analysis and immunofluorescence staining analysis. Cell counting kit-8, 5-ethynyl-2'-deoxyuridine and scratch wound assays were performed to assess the effects of hUSC-sEVs on the proliferation and migration ability of vaginal epithelial cells (VK2/E6E7). Quantitative real-time polymerase chain reaction (qRT-PCR) was carried out to test the expression of epithelial differentiation markers in VK2 cells. Moreover, a microRNA (miRNA) microarray was used to find hUSC-sEVs-specific miRNAs that potentially affected the proliferation, migration and differentiation ability of VK2 cells. RESULTS: The in vitro release profile revealed that the piGEL could ensure sustained release of hUSC-sEVs. The in vivo results showed that piGEL-sEVs effectively promoted epithelization and angiogenesis of vaginal mucosa defects in rabbits. According to miRNA microarray and qRT-PCR results, miR-126-3p might be the crucial molecule among the various miRNAs contained in hUSC-sEVs. The data showed that hUSC-sEVs promoted the migration and differentiation of VK2 cells by delivering miR-126-3p to suppress the expression of Spred1 and PIK3R2, thereby activating the ERK1/2 and ATK signaling pathways. CONCLUSION: The results indicated that piGEL-sEVs could be a novel promising approach for enhancing the epithelization of the neovagina after vaginoplasty and provided useful data for understanding the underlying mechanism of the effect of hUSC-sEVs on epithelization.

Kinetic and structural studies of the reaction of Escherichia coli dihydrodipicolinate synthase with (S)-2-bromopropionate.

Dihydrodipicolinate synthase (DHDPS) catalyzes the first committed step in the lysine-biosynthetic pathway converting pyruvate and L-aspartate-ß-semialdehyde to dihydrodipicolinate. Kinetic studies indicate that the pyruvate analog (S)-2-bromopropionate inactivates the enzyme in a pseudo-first-order process. An initial velocity pattern indicates that (S)-2-bromopropionate is a competitive inhibitor versus pyruvate, with an inhibition constant of about 8 mM. Crystals of DHDPS complexed with (S)-2-bromopropionate formed in a solution consisting of 50 mM HEPES pH 7.5, 18% polyethylene glycol 3350, 8 mM spermidine, 0.2 M sodium tartrate and 5.0 mg ml-1 DHDPS. The crystals diffracted to 2.15 Šresolution and belonged to space group P1. The crystal structure confirms the displacement of bromine and the formation of a covalent attachment between propionate and Lys161 at the active site of the enzyme. Lys161 is the active-site nucleophile that attacks the carbonyl C atom of pyruvate and subsequently generates an imine adduct in the first half-reaction of the ping-pong enzymatic reaction. A comparison of the crystal structures of DHDPS complexed with pyruvate or (S)-2-bromopropionate indicates the covalent adduct formed from (S)-2-bromopropionate leads to a rotation of about 180° of the ß-δ C atoms of Lys61 that aligns the covalently bound propionate fairly closely with the imine adduct formed with pyruvate.

Metabolomic Evaluation of N-Acetyl-p-Benzoquinone Imine Protein Adduct Formation with Therapeutic Acetaminophen Administration: Sex-based Physiologic Differences.

BACKGROUND: Acetaminophen (APAP)-associated transaminase elevation, induced by N-acetyl-p-benzoquinone imine (NAPQI) protein adduction, remains an area of research interest. Distinct from known genetic, physiologic, and dosage associations dictating severity of hepatic injury, no known factors predict an absence of protein adduct formation at therapeutic APAP dosing. HYPOTHESIS: Sex-based physiology is predictive of APAP-induced protein adduct formation and differential metabolite expression at therapeutic doses. METHODS: This retrospective study interrogated serum samples collected for a prior study investigating fluctuations of alanine aminotransferase (ALT) over time with 4G daily APAP dosing for ≥ 16 days in subjects from Denver, Colorado. Subjects were grouped by adduct formation (n = 184) vs no adducts (n = 20). Samples were run on ultra-high-performance liquid chromatography mass spectrometry from study days 0, 7, 16, and 31. Significant metabolite expressions were identified using t-tests with false discovery rate correction (FDR), partial least squares discriminant, and ANOVA simultaneous comparison analyses. Demographic and clinical data were explored using t-tests with FDR (age, weight, BMI, ALT) and Chi-square (sex, ethnicity, race) analyses. RESULTS: In pre-treatment samples, relative quantitation caprylic acid was expressed ninefold higher and 6-carboxyhexanoate was expressed threefold lower in subjects who did not develop adducts. Lactate had greater expression in the no adducts group (p = 0.001). Using absolute quantitation, glutathione was expressed 2.6-fold greater among no adduct subjects. Odds of males developing NAPQI protein adducts at therapeutic APAP dosing were 5.91 times lower than females (95% CI = 2.3-14.9; p = 0.0001). CONCLUSION: Multiple metabolites were differentially expressed based on adduct group and sex. Metabolites were identified unique to adduct development independent of sex. At therapeutic APAP dosing, males were less likely to develop APAP protein adducts. Further research into lipid biosynthesis and metabolism may provide further insight into physiology associated with adduct production.

An increase in surface hydrophobicity mediates chaperone activity in N-chlorinated RidA.

Under physiological conditions, Escherichia coli RidA is an enamine/imine deaminase, which promotes the release of ammonia from reactive enamine/imine intermediates. However, when modified by hypochlorous acid (HOCl), it turns into a potent chaperone-like holdase that can effectively protect E. coli's proteome during oxidative stress. However, it is unknown, which residues need to be chlorinated for activation. Here, we employ a combination of LC-MS/MS analysis, a chemo-proteomic approach, and a mutagenesis study to identify residues responsible for RidA's chaperone-like function. Through LC-MS/MS of digested RidAHOCl, we obtained direct evidence of the chlorination of one arginine residue. To overcome the instability of the N-chloramine modification, we established a chemoproteomic approach using 5-(dimethylamino) naphthalene-1-sulfinic acid (DANSO2H) as a probe to label N-chlorinated lysines. Using this probe, we were able to detect the N-chlorination of six additional lysine residues. Moreover, using a mutagenesis study to genetically probe the role of single arginine and lysine residues, we found that the removal of arginines R105 and/or R128 led to a substantial reduction of RidAHOCl's chaperone activity. These results, together with structural analysis, confirm that the chaperone activity of RidA is concomitant with the loss of positive charges on the protein surface, leading to an increased overall protein hydrophobicity. Molecular modelling of RidAHOCl and the rational design of a RidA variant that shows chaperone activity even in the absence of HOCl further supports our hypothesis. Our data provide a molecular mechanism for HOCl-mediated chaperone activity found in RidA and a growing number of other HOCl-activated chaperones.

Mitochondrial versus microsomal bioactivation of paracetamol by human liver and kidney tissues.

Mitochondria appeared to be a major target for paracetamol (PAR)-induced hepatotoxicity. Studies suggested that microsomal CYPs catalyse bioactivation of PAR to N-acetyl-p-benzoquinone imine (NAPQI), which alkylates mitochondrial proteins, and causes transmission of death signal from mitochondria to nucleus. We hypothesised that local formation of NAPQI within mitochondria seems more likely compared to the translocation of NAPQI. We therefore tested whether the formation of NAPQI may be catalysed by mitochondrial CYPs. Cellular fractions were isolated from human liver and kidney to compare the metabolic capacities. Liver and kidney mitochondria are capable to generate NAPQI. Mitochondrial CYP2E1 and CYP3A4 activities were comparable to the microsomal counterparts in both organs. Previously reported higher kidney microsomal CYP2E1 activity in men compared women were observed in mitochondrial CYP2E1 as well in the present study. On the other hand, no correlation between kidney CYP2E1 activity and quantity of NAPQI formation, as well as no induction on mitochondrial permeability transition pore (mPTP) opening by PAR in kidney mitochondria strongly suggested a different toxicity mechanism in this organ.

Development of an Efficient Extraction Method for Harvesting Gymnodimine-A from Large-Scale Cultures of Karenia selliformis.

Gymnodimine-A (GYM-A) is a fast-acting microalgal toxin and its production of certified materials requires an efficient harvesting technology from the large-scale cultures of toxigenic microalgae. In this study the recoveries of GYM-A were compared between several liquid-liquid extraction (LLE) treatments including solvents, ratios and stirring times to optimize the LLE technique for harvesting GYM-A from Karenia selliformis cultures, of which the dichloromethane was selected as the extractant and added to microalgal cultures at the ratio 55 mL L-1 (5.5%, v/v). The recovery of GYM-A obtained by the LLE technique was also compared with filtration and centrifugation methods. The stability of GYM-A in culture media were also tested under different pH conditions. Results showed that both the conventional filter filtration and centrifugation methods led to fragmentation of microalgal cells and loss of GYM-A in the harvesting processes. A total of 5.1 µg of GYM-A were obtained from 2 L of K. selliformis cultures with a satisfactory recovery of 88%. Interestingly, GYM-A obviously degraded in the culture media with the initial pH 8.2 and the adjusted pH of 7.0 after 7 days, but there was no obvious degradation in the acidic medium at pH 5.0. Therefore, the LLE method developed here permits the collection of large-volume cultures of K. selliformis and the high-efficiency extraction of GYM-A. This work provides a simple and valuable technique for harvesting toxins from large-scale cultures of GYM-producing microalgae.