Oxidative Biotransformation Research Articles

Discovery and characterization of NADH oxidases for selective sustainable synthesis of 5-hydroxymethylfuran carboxylic acid

Efficient regeneration of NAD+ remains a significant challenge for oxidative biotransformations. In order to identify enzymes with higher activity and stability, a panel of NADH oxidases (Nox) was investigated in the regeneration of nicotinamide cofactors for the oxidation of hydroxymethyl furfural (HMF) to 5-hydroxymethyl-2-furancarboxylic acid (HMFCA). We present novel Nox that exhibit remarkable catalytic activities, elevated thermal and pH stabilities, and higher intrinsic flavin loadings, thus eliminating the need for external flavin addition. The kinetic analysis of the NADH oxidases indicates that AdNox, GdNox, CmNox, and LvNox exhibit Vmax values of 86 U/mg, 50 U/mg, 4.3 U/mg, and 23 U/mg, respectively. When these NADH oxidases were applied in a HMF oxidation reaction, LvNox demonstrated the highest HMFCA yield of 97 % in the presence of 0.1 mM NAD and 10 mM HMF. In contrast to previously reported NADH oxidases from the same family, these NADH oxidases naturally accept NADPH as a substrate. Rapid kinetics experiments identified the oxidative reaction as the rate-limiting step of the reaction. NADH oxidases achieved high atom economy, a high reaction mass efficiency and a low process mass intensity. The findings contribute significantly to the field of biocatalysis and offer potential avenues for more environmentally friendly cofactor regeneration in chemical synthesis.

Concentration-dependent effects of the nerve agents cyclosarin and VX on cytochrome P450 in a HepaRG cell-based liver model.

The exposure to highly toxic organophosphorus (OP) compounds, including pesticides and nerve agents, is an ongoing medical challenge. OP can induce the uncontrolled overstimulation of the cholinergic system through inhibition of the enzyme acetylcholinesterase (AChE). The cytochrome P450 (CYP) enzymes in the liver play a predominant role in the metabolism of xenobiotics and are involved in the oxidative biotransformation of most clinical drugs. Previous research concerning the interactions between OP and CYP has usually focused on organothiophosphate pesticides that require CYP-mediated bioactivation to their active oxon metabolites to act as inhibitors of AChE. Since there has been little data available concerning the effect of nerve agents on CYP, we performed a study with cyclosarin (GF) and O-ethyl-S-[2-(diisopropylamino)-ethyl]-methylphosphonothioate (VX) by using a well-established, metabolically competent in vitro liver model (HepaRG cells). The inhibitory effect of the nerve agents GF and VX on the CYP3A4 enzyme was investigated showing a low CYP3A4 inhibitory potency. Changes on the transcription level of CYP and associated oxygenases were evaluated by quantitative reverse transcription polymerase chain reaction (qRT-PCR) using the two nerve agent concentrations 250 nM and 250 μM. In conclusion, the results demonstrated various effects on oxygenase-associated genes in dependence of the concentration and the structure of the nerve agent. Such information might be of relevance for potential interactions between nerve agents, antidotes or other clinically administered drugs, which are metabolized by the affected CYP, for example, for the therapy with benzodiazepines, that are used for the symptomatic treatment of OP poisoning and that require CYP-mediated biotransformation.

Oxidative Biotransformation of Organophosphotioate Pesticides and Acetylcholinesterase Enzymatic Inhibition

Introduction: Pesticides have lethal properties, capable of controlling or eliminating a living organism; they block the organisms' vital metabolic processes. They cause serious problems for human health, as they are highly toxic. The most used pesticides that are considered toxic are known as organophosphothioates (OP/P=S) in their commercialized form and organophosphates (OP/P=O) in their active form. These compounds have been the subject of studies on their metabolism and toxicology. According to research, these pesticides' toxicity is increased when oxidative metabolic desulfurization reactions occur, with the P=S bond being transformed into a P=O bond. This toxicity is due to the ability of OP/P=O species to inhibit the human acetylcholinesterase enzyme (HssAChE). Methods: To study the oxidative biotransformation of OP/P=S pesticides and the inhibition of the HssAChE enzyme by OP/P=S and OP/P=O using the molecular docking technique and QM/MM calculations. Results: The theoretical results showed that parathion is the compound with the greatest capacity to transform its P=S bonds into P=O bonds, thus forming the active paraoxon metabolite in the oxidative biotransformation process. In the HssAChE inhibition by OP/P=S and OP/P=O, our results showed that of all the compounds investigated, those with the highest inhibitory activities are parathion, paraoxon, malathion, diazoxon, chlorpyrifos and omethoate. Conclusion: This study was essential due to the lack of information in the literature about the oxidative biotransformation process of OP/P=S pesticides and the ability of these compounds to inhibit HssAChE. With this study, it was possible to observe that, in the oxidative biotransformation, chlorpyrifos and parathion have greater capacities to transform into their active metabolites and in the inhibition of the HssAChE enzyme, it was possible to observe that not all OF/P=O are the ones with the highest abilities to inhibit the HssAChE enzyme.

The Role of CYPs and Transporters in the Biotransformation and Transport of the Anti-hepatitis C Antiviral Agents Asunaprevir, Daclatasvir, and Beclabuvir: Impact of Liver Disease, Race and Drug-drug Interactions on Safety and Efficacy.

Asunaprevir, daclatasvir, and beclabuvir are direct-acting antiviral agents used in the treatment of patients infected with hepatitis C genotype 1b. This article reviews the biotransformation and disposition of these drugs in relation to the safety and efficacy of therapy. CYP3A4 and 3A5 catalyze the oxidative biotransformation of the drugs, while P-glycoprotein mediates their efflux from tissues. Asunaprevir is also a substrate for the influx transporters OATP1B1 and OATP2B1 and the efflux transporter MRP2, while beclabuvir is also a substrate for the efflux transporter BCRP. Liver disease decreases the expression of CYPs and transporters that mediate drug metabolism and disposition. Serum asunaprevir concentrations, but not those of daclatasvir or beclabuvir, are increased in patients with severe liver disease, which may produce toxicity. Pharmacogenomic variation in CYPs and transporters also has the potential to disrupt therapy with asunaprevir, daclatasvir and beclabuvir; some variants are more prevalent in certain racial groups. Pharmacokinetic drug-drug interactions, especially where asunaprevir, daclatasvir, and beclabuvir are victim drugs, are mediated by coadministered rifampicin, ketoconazole and ritonavir, and are attributable to inhibition and/or induction of CYPs and transporters. Conversely, there is also evidence that asunaprevir, daclatasvir and beclabuvir are perpetrators of drug interactions with coadministered rosuvastatin and dextromethorphan. Together, liver disease, pharmacogenomic variation and drug-drug interactions may disrupt therapy with asunaprevir, daclatasvir and beclabuvir due to the impaired function of important CYPs and transporters.

Bioaccumulation and biotransformation of plasticisers diisononyl phthalate and di(2-ethylhexyl) terephthalate in black soldier fly larvae reared on (micro)plastic-contaminated food waste

Food waste is currently used for the production of biogas. However, a reusage of waste is preferred to follow the principles of the circular economy and consider the waste management hierarchy, which can be achieved by rearing black soldier fly (BSF) larvae on such organic waste. Nonetheless, the presence of (micro)plastics and related additive plasticisers might induce chemical safety hazards to the larval applications as feed. Therefore, the bioaccumulation and biotransformation of two plasticisers (diisononyl phthalate (DINP) and di(2-ethylhexyl) terephthalate (DEHT)) in BSF larvae reared on food waste streams contaminated with polyvinyl chloride (PVC) (micro)plastics were investigated. Results showed that BSF larvae appeared to have a moderate intake of DINP during its rearing phase of 10 days (82 - 273 ng/g), while being able to biotransform it into the primary biotransformation product monoisononyl phthalate (MINP) within 24 h. For DEHT, an uptake of 67 - 137 ng/g was measured in the BSF larvae; however, no clear biotransformation pattern was observed. In addition, while no secondary oxidative biotransformation products were found in the larvae, these were measured in the frass, leading to the hypothesis that microorganism-mediated biotransformation of plasticisers occurred. In conclusion, based on the results of this study, BSF larvae could potentially be used safely in the frame of circular economy, when reared on organic substrates contaminated with the same PVC microplastic content and sizes used.

Tissue Distribution and Metabolization of Ciguatoxins in an Herbivorous Fish following Experimental Dietary Exposure to Gambierdiscus polynesiensis.

Ciguatoxins (CTXs), potent neurotoxins produced by dinoflagellates of the genera Gambierdiscus and Fukuyoa, accumulate in commonly consumed fish species, causing human ciguatera poisoning. Field collections of Pacific reef fish reveal that consumed CTXs undergo oxidative biotransformations, resulting in numerous, often toxified analogs. Following our study showing rapid CTX accumulation in flesh of an herbivorous fish, we used the same laboratory model to examine the tissue distribution and metabolization of Pacific CTXs following long-term dietary exposure. Naso brevirostris consumed cells of Gambierdiscus polynesiensis in a gel food matrix over 16 weeks at a constant dose rate of 0.36 ng CTX3C equiv g-1 fish d-1. CTX toxicity determination of fish tissues showed CTX activity in all tissues of exposed fish (eight tissues plus the carcass), with the highest concentrations in the spleen. Muscle tissue retained the largest proportion of CTXs, with 44% of the total tissue burden. Moreover, relative to our previous study, we found that larger fish with slower growth rates assimilated a higher proportion of ingested toxin in their flesh (13% vs. 2%). Analysis of muscle extracts revealed the presence of CTX3C and CTX3B as well as a biotransformed product showing the m/z transitions of 2,3-dihydroxyCTX3C. This is the first experimental evidence of oxidative transformation of an algal CTX in a model consumer and known vector of CTX into the fish food web. These findings that the flesh intended for human consumption carries the majority of the toxin load, and that growth rates can influence the relationship between exposure and accumulation, have significant implications in risk assessment and the development of regulatory measures aimed at ensuring seafood safety.

Mass Balance and Metabolic Pathways of Eliapixant, a P2X3 Receptor Antagonist, in Healthy Male Volunteers.

Overactive adenosine triphosphate signaling via P2X3 homotrimeric receptors is implicated in multiple conditions. To fully understand the metabolism and elimination pathways of eliapixant, a study was conducted to assess the pharmacokinetics, mass balance, and routes of excretion of a single oral dose of the selective P2X3 receptor antagonist eliapixant, in addition to an invitro characterization. In this single-center open-label non-randomized non-placebo-controlled phase I study, healthy male subjects (n=6) received a single dose of 50mg eliapixant blended with 3.7MBq [14C]eliapixant as a PEG 400-based oral solution. Total radioactivity and metabolites excreted in urine and feces, and pharmacokinetics of total radioactivity, eliapixant, and metabolites in plasma were assessed via liquid scintillation counting and high-performance liquid chromatography-based methods coupled to radiometric and mass spectrometric detection. Metabolite profiles of eliapixant in human invitro systems and metabolizing enzymes were also investigated. After administration as an oral solution, eliapixant was rapidly absorbed, reaching maximum plasma concentrations within 2h. Eliapixant was eliminated from plasma with a mean terminal half-life of 48.3h. Unchanged eliapixant was the predominant component in plasma (72.6% of total radioactivity area under the curve). The remaining percentage of drug-related components in plasma probably represented the sum of many metabolites, detected in trace amounts. Mean recovery of total radioactivity was 97.9% of the administered dose (94.3-99.4%) within 14 days, with 86.3% (84.8-88.1%) excreted via feces and 11.6% (9.5-13.1%) via urine. Excretion of parent drug was minimal in feces (0.7% of dose) and urine (≈0.5%). In feces, metabolites formed by oxidation represented > 90% of excreted total radioactivity. The metabolites detected in the invitro experiments were similar to those identified invivo. Complete recovery of administered eliapixant-related radioactivity was observed in healthy male subjects with predominant excretion via feces. Eliapixant was almost exclusively cleared by oxidative biotransformation (>90% of dose), with major involvement of cytochrome P450 3A4. Excretion of parent drug was of minor importance (~1% of dose). ClinicalTrials.gov: NCT04487431 (registered 27 July 2020)/EudraCT number: 2020-000519-54 (registered 3 February 2020), NCT02817100 (registered 26 June 2016), NCT03310645 (registered 16 October 2017).

Expected and Unexpected Products from the Biochemical Oxidation of Bacterial Alkylquinolones with CYP4F11.

2-Alkylquinolones are a class of microbial natural products primarily produced in the Pseudomonas and Burkholderia genera that play a key role in modulating quorum sensing. Bacterial alkylquinolones were synthesized and then subjected to oxidative biotransformation using human cytochrome P450 enzyme CYP4F11, heterologously expressed in the fission yeast Schizosaccharomyces pombe. This yielded a range of hydroxylated and carboxylic acid derivatives which had undergone ω-oxidation of the 2-alkyl chain, the structures of which were determined by analysis of NMR and MS data. Oxidation efficiency depended on chain length, with a chain length of eight or nine carbon atoms proving optimal for high yields. Homology modeling suggested that Glu233 was relevant for binding, due to the formation of a hydrogen bond from the quinolone nitrogen to Glu233, and in this position only the longer alkyl chains could come close enough to the heme moiety for effective oxidation. In addition to the direct oxidation products, a number of esters were also isolated, which was attributed to the action of endogenous yeast enzymes on the newly formed ω-hydroxy-alkylquinolones. ω-Oxidation of the alkyl chain significantly reduced the antimicrobial and antibiofilm activity of the quinolones.

Pharmacokinetic Interactions Between Antiseizure and Psychiatric Medications.

Antiseizure medications and drugs for psychiatric diseases are frequently used in combination. In this context, pharmacokinetic interactions between these drugs may occur. The vast majority of these interactions are primarily observed at a metabolic level and result from changes in the activity of the cytochrome P450 (CYP). Carbamazepine, phenytoin, and barbiturates induce the oxidative biotransformation and can consequently reduce the plasma concentrations of tricyclic antidepressants, many typical and atypical antipsychotics and some benzodiazepines. Newer antiseizure medications show a lower potential for clinically relevant interactions with drugs for psychiatric disease. The pharmacokinetics of many antiseizure medications is not influenced by antipsychotics and anxiolytics, while some newer antidepressants, namely fluoxetine, fluvoxamine and viloxazine, may inhibit CYP enzymes leading to increased serum concentrations of some antiseizure medications, including phenytoin and carbamazepine. Clinically relevant pharmacokinetic interactions may be anticipated by knowledge of CYP enzymes involved in the biotransformation of individual medications and of the influence of the specific comedication on the activity of these CYP enzymes. As a general rule, these interactions can be managed by careful evaluation of clinical response and, when indicated, individualized dosage adjustments guided by measurement of drugs serum concentrations, especially if pharmacokinetic interactions may cause any change in seizure control or signs of toxicity. Further studies are required to improve predictions of pharmacokinetic interactions between antiseizure medications and drugs for psychiatric diseases providing practical helps for clinicians in the clinical setting.

In vitro cannabinoid receptor activity, metabolism, and detection in seized samples of CH-PIATA, a new indole-3-acetamide synthetic cannabinoid.

The rapidly evolving synthetic cannabinoid receptor agonist (SCRA) market poses significant challenges for forensic scientists. Since the enactment of a generic ban in China, a variety of new compounds have emerged capable of evading the legislation by carrying new structural features. One recent example of a SCRA with new linker and head moieties is CH-PIATA (CH-PIACA, CHX-PIATA, CHX-PIACA). CH-PIATA bears an additional methylene spacer in the linker moiety between the indole core and the traditional carbonyl component of the linker. This study describes detections in 2022 of this new SCRA in the United States, Belgium, and Scottish prisons. CH-PIATA was detected once in a seized powder by Belgian customs and 12 times in Scottish prisons in infused papers or resin. The metabolites of CH-PIATA were investigated via in vitro human liver microsome (HLM) incubations and eight metabolites were identified, dominated by oxidative biotransformations. A blood sample from the United States was confirmed to contain a mixture of SCRAs including CH-PIATA via presence of the parent and at least five of the metabolites identified from HLM incubations. Furthermore, this paper evaluates the intrinsic in vitro cannabinoid 1 and 2 (CB1 and CB2) receptor activation potential of CH-PIATA reference material and the powder seized by Belgian customs by means of β-arrestin 2 recruitment assays. Both the reference and the seized powder showed a weak activity at both CB receptors with signs of antagonism found. Based on these results, the expected harm potential of this newly emerging substance remains limited.

Metabolism and Disposition of the Novel Oral Factor XIa Inhibitor Asundexian in Rats and in Humans.

Current anticoagulants pose an increased risk of bleeding. The development of drugs targeting factor XIa, like asundexian, may provide a safer treatment option. A human mass‑balance study was conducted to gain a deeper understanding of the absorption, distribution, metabolism, excretion, and potential for drug-drug interaction of asundexian. Additionally, an overview of the biotransformation and clearance pathways for asundexian in humans and bile-duct cannulated (BDC) rats in vivo, as well as in vitro in hepatocytes of both species, is reported. The mass balance, biotransformation, and excretion pathways of asundexian were investigated in six healthy volunteers (single oral dose of 25 mg [14C]asundexian) and in BDC rats (intravenous [14C]asundexian 1 mg/kg). Overall recovery of radioactivity was 101% for humans (samples collected up to 14 days after dosing), and 97.9% for BDC rats (samples collected in the 24 h after dosing). Radioactivity was mainly excreted into feces in humans (80.3%) and into bile/feces in BDC rats (> 94%). The predominant clearance pathways in humans were amide hydrolysis to metabolite M1 (47%) and non-labeled M9 with subsequent N-acetylation to M10; oxidative biotransformation was a minor pathway (13%). In rats, hydrolysis of the terminal amide to M2 was the predominant pathway. In human plasma, asundexian accounted for 61.0% of total drug-related area under the plasma concentration-time curve (AUC); M10 was the major metabolite (16.4% of the total drug-related AUC). Excretion of unmetabolized drug was a significant clearance pathway in both species (human, ~ 37%; BDC rat, ~ 24%). The near-complete bioavailability of asundexian suggests negligible limitations on absorption and first-pass metabolism. Comparison with radiochromatograms from incubations with human or rat hepatocytes indicated consistency across species and a good overall in vitro/in vivo correlation. Similar to preclinical experiments, total asundexian-derived radioactivity is cleared quantitatively predominantly via feces. Excretion occurs mainly via amide hydrolysis and as the unchanged drug.

Biotransformation of ethylene glycol to glycolic acid by Yarrowia lipolytica: a route for poly(ethylene terephthalate) (PET) upcycling.

Biological recycling of PET waste has been extensively investigated recently to tackle plastic waste pollution, and ethylene glycol (EG) is one of the main building blocks recovered from this process. Wild-type Yarrowia lipolytica IMUFRJ 50682 can be used as biocatalyst for this purpose. Herein, we report its ability to perform oxidative biotransformation of EG into glycolic acid (GA): a higher value-added chemical with varied industrial applications. Maximum non-inhibitory concentration assay for EG cytotoxicity revealed that this yeast tolerates high concentrations (up to 2 M). Whole-cell biotransformation assays using resting yeast cells showed GA production uncoupled to cell growth metabolism, and 13 C nuclear magnetic resonance (NMR) analysis confirmed GA production. Moreover, higher agitation speed (450 versus 350 rpm) resulted in a 1.12-fold GA production improvement (from 352 to 429.5 mM) during Y. lipolytica cultivation in bioreactors after 72 h. GA was constantly accumulated in the medium, suggesting that this yeast may also share an incomplete oxidation pathway (i.e., it is not metabolized to carbon dioxide) as seen in acetic acid bacterial group. Additional assays using higher chain-length diols (1,3-propanediol, 1,4-butanediol, and 1,6-hexanediol) revealed that C4 and C6 diols were more cytotoxic, suggesting that they underwent different pathways in the cells. We found that this yeast consumed extensively all these diols, however, 13 C NMR analysis from supernatant identified solely the presence of 4-hydroxybutanoic acid from 1,4-butanediol, along with GA from EG oxidation. Findings reported herein reveal a potential route for PET upcycling to a higher value-added product. This article is protected by copyright. All rights reserved.

<i>Rhodococcus rhodochrous</i> IEGM 1360, an Efficient Biocatalyst of C3 Oxidative Transformation of Oleanane Triterpenoids

Abstract—The optimal conditions for C3 oxidative biotransformation of 1.0 g/L pentacyclic triterpenoids oleanolic (OA) and glycyrrhetic (GA) acids were determined using the resting cells of Rhodococcus rhodochrous IEGM 1360 from the Regional Specialised Collection of Alkanotrophic Microorganisms. Suspensions of the resting cells with OD600 2.6 in pH 8.0 buffer and OD600 2.2 in pH 6.0 buffer showed the highest catalytic activity against OA and GA, which ensured the formation of 61 and 100% of their 3-oxo derivatives, respectively. Using phase contrast, atomic force, and confocal laser scanning microscopy, an adaptive response of rhodococci to the effects of OA and GA was revealed. In silico, the apoptotic and antioxidant activities of 3-oxo-OA and 3-oxo-GA, respectively, have been assumed. In vitro, a pronounced antibacterial activity of 3-oxo-OA against Micrococcus luteus, Escherichia coli, Staphylococcus aureus, and Bacillus subtilis was shown. The absence of toxic effects of the triterpenoids and their 3-oxo derivatives on aquatic and plant objects was demonstrated in silico and in vitro, respectively.

NADPH–cytochrome P450 reductase knockdown decreases the response to precocene I in the migratory locust Locusta migratoria

Precocene I is a juvenile hormone antagonist that needs to be activated via oxidative biotransformation catalyzed by cytochrome P450 (CYP). NADPH–cytochrome P450 reductase (CPR) supplies CYP with electrons in the oxidation–reduction process; however, its functional role in the activation of precocene I remains unexplored. Here, the representative characteristics of CPRs were analyzed in the CPR gene of Locusta migratoria (LmCPR), the result of model docking indicated that the hydrogen bonds were formed between reduced nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN) and NADPH-, FAD-, FMN-domains of LmCPR, respectively. Treating the fourth-instar nymphs with precocene I decreased the juvenile hormone titers of nymphs to 0.55-fold of that in acetone-treated controls, and extended the interval time between fourth- and fifth-instar nymphs. 68.75% of the treated fourth-instar nymphs developed into precocious adults in the fifth-instar. LmCPR knockdown decreased the response to precocene I in the nymphs, the occurrence rate of precocious adults induced by precocene I treatment reduced by 23.11%. Therefore, LmCPR may be involved in the activation of precocene I in L. migratoria. In addition, we generated an active recombinant LmCPR protein using a prokaryotic expression system, its activity in reducing cytochrome c was 33.13 ± 11.50 nmol CytCred/min/μg protein. This study lays the foundation for further research on the role of LmCPR in precocene I activation.

A pilot study for enhanced transformation of a metabolite 3,5-dichloroaniline derived from dicarboximide fungicides through immobilized laccase mediator system.

This pilot investigation aimed to evaluate the removal efficiency and the underlying biocatalytic pathways of immobilized fungal laccase during the oxidative biotransformation of a non-phenolic metabolite, 3,5-dichloroaniline (3,5-DCA) derived from dicarboximide fungicides. The maximum loading of laccase on the microporous support surfaces could reach 36.4mg/g. The immobilized laccase on the microporous support surfaces exhibited excellent thermal stability, pH adaptability, storage stability, and reusability compared to free laccase. The ILMS assay indicated that the immobilized laccase efficiently removed studied 3,5-DCA (99-100%) in the aqueous medium, within 72h in the presence of catechol. In this study, we identified three coupling reaction products during the removal of 3,5-DCA through an ILMS assay. Based on the identified coupling reaction products, we proposed the reaction pathway for the biotransformation of 3,5-DCA by immobilized laccase, which was shown to be potentially useful in the sustainable environmental remediation of aniline metabolite (i.e., 3,5-DCA) derived from dicarboximide fungicides.

Multiwell photocatalytic microreactor device integrating drug biotransformation modeling and sample preparation on a MALDI target

Metabolic conversion of drugs to chemically reactive products, known as bioactivation, is considered to underlie the adverse drug reactions such as idiosyncratic hepatotoxicity. As bioactivation of most drugs involves phase I oxidation, UV-induced TiO2 photocatalytic oxidation (UV/TiO2-PCO) can be used as a fast, simple and inexpensive non-enzymatic method of oxidative metabolism simulation allowing for rapid preliminary screening of candidate drugs for reactive metabolite formation. We developed and evaluated a novel prototype 96-well on-target UV/TiO2-photocatalytic microreactor setup (PCμR96) that integrates drug metabolism simulation and sample preparation directly on a MALDI target. PCμR96 allows to sequentially perform UV/TiO2-PCO of xenobiotics, GSH or protein adduct formation, protein digestion and sample concentration, followed by surface- or matrix-assisted laser desorption ionization (SALDI/MALDI) mass spectrometry (MS). Amodiaquine (AQ), a well-studied antimalarial drug that is known to undergo oxidative biotransformation, was selected as a model compound. Oxidation products of AQ and their protein adducts were obtained using PCμR96 and identified by SALDI and MALDI MS/MS analysis. The achieved AQ oxidation products were in good agreement with the known AQ metabolites produced in biological systems. Two of the products were proved to be reactive towards cysteyl thiols of human globin.

Molecular insights into the unusually promiscuous and catalytically versatile Fe(II)/\u03b1-ketoglutarate-dependent oxygenase SptF

Non-heme iron and α-ketoglutarate-dependent (Fe/αKG) oxygenases catalyze various oxidative biotransformations. Due to their catalytic flexibility and high efficiency, Fe/αKG oxygenases have attracted keen attention for their application as biocatalysts. Here, we report the biochemical and structural characterizations of the unusually promiscuous and catalytically versatile Fe/αKG oxygenase SptF, involved in the biosynthesis of fungal meroterpenoid emervaridones. The in vitro analysis revealed that SptF catalyzes several continuous oxidation reactions, including hydroxylation, desaturation, epoxidation, and skeletal rearrangement. SptF exhibits extremely broad substrate specificity toward various meroterpenoids, and efficiently produced unique cyclopropane-ring-fused 5/3/5/5/6/6 and 5/3/6/6/6 scaffolds from terretonins. Moreover, SptF also hydroxylates steroids, including androsterone, testosterone, and progesterone, with different regiospecificities. Crystallographic and structure-based mutagenesis studies of SptF revealed the molecular basis of the enzyme reactions, and suggested that the malleability of the loop region contributes to the remarkable substrate promiscuity. SptF exhibits great potential as a promising biocatalyst for oxidation reactions.

An insight into an intriguing oxidative biotransformation pathway of 5-O-caffeoylquinic acid by a gut bacterium.

Microbiota is known to play a pivotal role in generating bioavailable and bioactive low-molecular-weight metabolites from dietary polyphenols. 5-O-caffeoylquinic acid (5-CQA), one of the main polyphenols found in human diet, was submitted to a resting cell biotransformation study using three gut bacteria species Lactobacillus reuteri, Bacteroides fragilis and Bifidobacterium longum. These bacteria were selected according to their belonging to the main phyla found in human gut microbiota. Our study highlighted the ability of only one of the strains studied, L. reuteri, to bioconverse 5-CQA into various metabolites due to the expression of the cinnamoyl esterase enzyme as the first step. Interestingly, one known natural compound, esculetin, was described for the first time as a 5-CQA-derived metabolite after conversion by a gut bacterium, the other metabolites had already been reported. This evidence highlighted an interesting oxidative pathway occurring in vivo by intestinal microbiota leading to esculetin. This molecule was also identified after electrochemical and enzymatic oxidations of caffeic acid. The oxidation capacity of L. reuteri led to less diverse metabolites in comparison to those obtained either electrochemically and enzymatically where dimers and trimers were reported. Thus, esculetin may have interesting and benefical biological effects on gut microbiota, which should be further evaluated. Novel synbiotics could be formulated from the association of L. reuteri with 5-CQA.

Production and characterization of a novel thermostable laccase from Bacillus licheniformis VNQ and its application in synthesis of bioactive 1,4-naphthoquinones

Bacterial laccases have proven to be a potential biocatalyst for various industrial applications due to their remarkable catalytic and stability properties. In this study, a novel thermostable laccase was produced from the bacterium Bacillus licheniformis VNQ by submerged fermentation. The specific activity of crude and purified laccase was found to be 13.17U mg-1 and 83.47U mg-1, respectively. The enzyme possessed a molecular mass of ∼48kDa when characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The optimum temperature and pH for enzyme activity was determined to be 55°C and 5.0, respectively. The enzyme was considered to be thermo-tolerant as it possessed a half-life of 4h at 70°C. The enzyme was utilized for the oxidative biotransformation of in situ synthesized p-quinones to biologically active compounds, 1,4-naphthoquinone and its derivative. The obtained products were characterized using nuclear magnetic resonance (NMR) spectroscopy and gas chromatography-mass spectrometry (GC-MS) analysis. A high yield of naphthoquinones (74.93±1.2%) with 1,4-naphthoquinone (60.61±1.0%), and its derivative 2-hydroxy-1,4-naphthoquinone (14.32±0.2%) was obtained at the optimized reaction conditions.

FNR-Type Regulator GoxR of the Obligatorily Aerobic Acetic Acid Bacterium Gluconobacter oxydans Affects Expression of Genes Involved in Respiration and Redox Metabolism.

Gene expression in the obligately aerobic acetic acid bacterium Gluconobacter oxydans responds to oxygen limitation, but the regulators involved are unknown. In this study, we analyzed a transcriptional regulator named GoxR (GOX0974), which is the only member of the fumarate-nitrate reduction regulator (FNR) family in this species. Evidence that GoxR contains an iron-sulfur cluster was obtained, suggesting that GoxR functions as an oxygen sensor similar to FNR. The direct target genes of GoxR were determined by combining several approaches, including a transcriptome comparison of a ΔgoxR mutant with the wild-type strain and detection of in vivo GoxR binding sites by chromatin affinity purification and sequencing (ChAP-Seq). Prominent targets were the cioAB genes encoding a cytochrome bd oxidase with low O2 affinity, which were repressed by GoxR, and the pnt operon, which was activated by GoxR. The pnt operon encodes a transhydrogenase (pntA1A2B), an NADH-dependent oxidoreductase (GOX0313), and another oxidoreductase (GOX0314). Evidence was obtained for GoxR being active despite a high dissolved oxygen concentration in the medium. We suggest a model in which the very high respiration rates of G. oxydans due to periplasmic oxidations cause an oxygen-limited cytoplasm and insufficient reoxidation of NAD(P)H in the respiratory chain, leading to inhibited cytoplasmic carbohydrate degradation. GoxR-triggered induction of the pnt operon enhances fast interconversion of NADPH and NADH by the transhydrogenase and NADH reoxidation by the GOX0313 oxidoreductase via reduction of acetaldehyde formed by pyruvate decarboxylase to ethanol. In fact, small amounts of ethanol were formed by G. oxydans under oxygen-restricted conditions in a GoxR-dependent manner.IMPORTANCEGluconobacter oxydans serves as a cell factory for oxidative biotransformations based on membrane-bound dehydrogenases and as a model organism for elucidating the metabolism of acetic acid bacteria. Surprisingly, to our knowledge none of the more than 100 transcriptional regulators encoded in the genome of G. oxydans has been studied experimentally until now. In this work, we analyzed the function of a regulator named GoxR, which belongs to the FNR family. Members of this family serve as oxygen sensors by means of an oxygen-sensitive [4Fe-4S] cluster and typically regulate genes important for growth under anoxic conditions by anaerobic respiration or fermentation. Because G. oxydans has an obligatory aerobic respiratory mode of energy metabolism, it was tempting to elucidate the target genes regulated by GoxR. Our results show that GoxR affects the expression of genes that support the interconversion of NADPH and NADH and the NADH reoxidation by reduction of acetaldehyde to ethanol.