One-Pot Synthesis of Useful S-Substituted-l-cysteine Sulfoxides Using Genetically Engineered Escherichia coli.

S-Substituted-l-cysteine sulfoxides are valuable compounds that are contained in plants. Particularly, (+)-alliin and its degraded products have gained significant attention because of their human health benefits. However, (+)-alliin production has been limited to extraction from plants and chemical synthesis; both methods have drawbacks in terms of stability and safety. Here, we proposed the enzymatic cascade reaction for synthesizing (+)-alliin from readily available substrates. To achieve a one-pot (+)-alliin production, we constructed Escherichia coli coexpressing the genes encoding tryptophan synthase from Aeromonas hydrophila ssp. hydrophila NBRC 3820 and l-isoleucine hydroxylase from Bacillus thuringiensis 2e2 for the biocatalyst. Deletion of tryptophanase gene in E. coli increased the yield about 2-fold. Under optimized conditions, (+)-alliin accumulation reached 110 mM, which is the highest productivity thus far. Moreover, natural and unnatural S-substituted-l-cysteine sulfoxides were synthesized by applying various thiols to the cascade reaction. These results indicate that the developed bioprocess would enable the supply of diverse S-substituted-l-cysteine sulfoxides.

Acidification and tissue disruption affect glucosinolate and S-methyl-l-cysteine sulfoxide hydrolysis and formation of amines, isothiocyanates and other organosulfur compounds in red cabbage (Brassica oleracea var. capitata f. rubra).

Cabbages are rich in sulfur-containing metabolites like glucosinolates (GLSs) and S-methyl-l-cysteine sulfoxide (SMCSO). Tissue disruption initiates hydrolysis of these compounds and bioactive volatile hydrolysis products such as isothiocyanates (ITCs), sulfides, and thiosulfinates are formed. However, nitriles, epithionitriles, or amines can also result from GLSs. Here, the influence of hydrolysis time, extent of tissue disruption (chopping vs. homogenization), and addition of lemon juice or vinegar on the outcome of enzymatic hydrolysis of GLSs and SMCSO was investigated in red cabbage. Chopping led to partial hydrolysis of GLSs, whereas homogenization completely degraded GLSs but only had a small effect on SMCSO. Homogenization increased amine formation from alkenyl and methylthioalkyl ITCs, but not from methylsulfinylalkyl ITCs. Acidification inhibited formation of products from SMCSO. Further, it reduced nitrile and epithionitrile formation and stopped amine formation, thereby increasing ITC levels. Therefore, acidification is a valuable mean to enhance ITC levels in fresh Brassica foods.

Directed evolution of Baeyer-Villiger monooxygenase for highly secretory expressed in Pichia pastoris and efficient preparation of chiral pyrazole sulfoxide.

The methylotrophic yeast Pichia pastoris (Komagataella phaffii) is a highly distinguished expression platform for the excellent synthesis of various heterologous proteins in recent years. With the advantages of high-density fermentation, P. pastoris can produce gram amounts of recombinant proteins. While not every protein of interest can be expressed to such high titers, such as Baeyer-Villiger monooxygenase (BVMO) (AcPSMO) which is responsible for pyrazole sulfide asymmetric oxidation. In this work, an excellent yeast expression system was established to facilitate efficient AcPSMO expression, which exhibited 9.5-fold enhanced secretion. Subsequently, an ultrahigh throughput screening method based on fluorescence-activated cell sorting by fusing super folder green fluorescent protein (sfGFP) in the C-terminal of AcPSMO was developed, and directed evolution was performed. The protein expression level of the superior mutant AcPSMOP1 (S58T/T252P/E336N/H456D) reached 84.6 mg/L at 100 mL shaking flask, which was 4.7 times higher than the levels obtained with the wild-type. Finally, the optimized chassis cells were used for high-density fermentation on a 5-L scale, and AcPSMOP1 protein yield of 3.4 g/L was achieved, representing approximately 85% of the total protein secreted. By directly employing the pH-adjusted supernatant as a biocatalyst, 20 g/L pyrmetazole sulfide was completely transformed into the corresponding (S)-sulfoxide, with a 78.8% isolated yield. This work confers dramatic benefits for efficient secretion of other BVMOs in P. pastoris.

Sulforaphane and Sulforaphane-Nitrile Metabolism in Humans Following Broccoli Sprout Consumption: Inter-individual Variation, Association with Gut Microbiome Composition, and Differential Bioactivity.

SCOPE: The glucosinolate glucoraphanin from broccoli is converted to sulforaphane (SFN) or sulforaphane-nitrile (SFN-NIT) by plant enzymes or the gut microbiome. Human feeding studies typically observe high inter-individual variation in absorption and excretion of SFN, however, the source of this variation is not fully known. To address this, a human feeding trial to comprehensively evaluate inter-individual variation in the absorption and excretion of all known SFN metabolites in urine, plasma, and stool, and tested the hypothesis that gut microbiome composition influences inter-individual variation in total SFN excretion has been conducted. METHODS AND RESULTS: Participants (n = 55) consumed a single serving of broccoli or alfalfa sprouts and plasma, stool, and total urine are collected over 72 h for quantification of SFN metabolites and gut microbiome profiling using 16S gene sequencing. SFN-NIT excretion is markedly slower than SFN excretion (72 h vs 24 h). Members of genus Bifidobacterium, Dorea, and Ruminococcus torques are positively associated with SFN metabolite excretion while members of genus Alistipes and Blautia has a negative association. CONCLUSION: This is the first report of SFN-NIT metabolite levels in human plasma, urine, and stool following consumption of broccoli sprouts. The results help explain factors driving inter-individual variation in SFN metabolism and are relevant for precision nutrition.

Characterization of phytochemicals from twisted-leaf garlic (Allium obliquum L.) using liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry.

INTRODUCTION: Twisted-leaf garlic (Allium obliquum L.) is a wild Allium species, which is traditionally used as aroma plant for culinary purposes due to its unique, garlic-like flavor. It represents an interesting candidate for domestication, breeding and cultivation. OBJECTIVES: The objective of this work was to explore and comprehensively characterize polar and semi-polar phytochemicals accumulating in leaves and bulbs of A. obliquum. METHOD: Plant material obtained from a multiyear field trial was analyzed using a metabolite profiling workflow based on ultra-high performance liquid chromatography-coupled electrospray ionization quadrupole time-of-flight mass spectrometry (UHPLC/ESI-QTOFMS) and two chromatographic methods. For annotation of metabolites, tandem mass spectrometry experiments were carried out and the resulting accurate-mass collision-induced dissociation (CID) mass spectra interpreted. Onion and garlic bulb extracts were used as reference samples. RESULTS: Important metabolite classes influencing nutritional, sensory and technological properties were detected and structurally characterized including fructooligosaccharides with a degree of polymerization of 3-5, S-alk(en)ylcysteine sulfoxides and other S-substituted cysteine conjugates, flavonoids including O- and C-glycosylated flavones as well as O-glycosylated flavonols, steroidal saponins, hydroxycinnamic acid conjugates, phenylethanoids and free sphingoid bases. In addition, quantitative data for non-structural carbohydrates, S-alk(en)ylcysteine sulfoxides and flavonoids are provided. CONCLUSION: The compiled analytical data including CID mass spectra of more than 160 annotated metabolites provide for the first time a phytochemical inventory of A. obliquum and lay the foundation for its further use as aroma plant in food industry.

Methionine sulfoxide reductases and cholesterol transporter STARD3 constitute an efficient system for detoxification of cholesterol hydroperoxides.

Methionine sulfoxide reductases (MSRs) are key enzymes in the cellular oxidative defense system. Reactive oxygen species oxidize methionine residues to methionine sulfoxide, and the methionine sulfoxide reductases catalyze their reduction back to methionine. We previously identified the cholesterol transport protein STARD3 as an in vivo binding partner of MSRA (methionine sulfoxide reductase A), an enzyme that reduces methionine-S-sulfoxide back to methionine. We hypothesized that STARD3 would also bind the cytotoxic cholesterol hydroperoxides and that its two methionine residues, Met307 and Met427, could be oxidized, thus detoxifying cholesterol hydroperoxide. We now show that in addition to binding MSRA, STARD3 binds all three MSRB (methionine sulfoxide reductase B), enzymes that reduce methionine-R-sulfoxide back to methionine. Using pure 5, 6, and 7 positional isomers of cholesterol hydroperoxide, we found that both Met307 and Met427 on STARD3 are oxidized by 6α-hydroperoxy-3ß-hydroxycholest-4-ene (cholesterol-6α-hydroperoxide) and 7α-hydroperoxy-3ß-hydroxycholest-5-ene (cholesterol-7α-hydroperoxide). MSRs reduce the methionine sulfoxide back to methionine, restoring the ability of STARD3 to bind cholesterol. Thus, the cyclic oxidation and reduction of methionine residues in STARD3 provides a catalytically efficient mechanism to detoxify cholesterol hydroperoxide during cholesterol transport, protecting membrane contact sites and the entire cell against the toxicity of cholesterol hydroperoxide.

Metabolomic and transcriptomic analysis of cold plasma promoting biosynthesis of active substances in broccoli sprouts.

INTRODUCTION: Broccoli sprouts have great health and commercial value because they are rich in sulforaphane, a special bioactive compound that helps to prevent chronic diseases, such as cancer and cardiovascular disease. OBJECTIVE: The aim of this study was to increase the levels of active substances in broccoli sprouts and understand their metabolic mechanisms. METHODOLOGY: Metabolomics based on liquid chromatography-tandem mass spectrometry and transcriptome analysis were combined to analyse the enrichment of metabolites in broccoli sprouts treated with cold plasma. RESULTS: After 2 min of cold plasma treatment, the contents of sulforaphane, glucosinolates, total phenols, and flavonoids, as well as myrosinase activity, were greatly improved. Transcriptomics revealed 7460 differentially expressed genes in the untreated and treated sprouts. Metabolomics detected 6739 differential metabolites, including most amino acids, their derivatives, and organic acids. Enrichment analyses of metabolomics and transcriptomics identified the 20 most significantly differentially expressed metabolic pathways. CONCLUSIONS: Overall, cold plasma treatment can induce changes in the expression and regulation of certain metabolites and genes encoding active substances in broccoli sprouts.

Proteome reveals the mechanism of selenium-sulfur interaction in regulating isothiocyanate biosynthesis and the physiological metabolism of broccoli sprouts.

Broccoli sprouts have a strong ability to accumulate isothiocyanate and selenium. In this study, the isothiocyanate content increased significantly as a result of ZnSO4 stress. Particularly, based on the isothiocyanate content is not affected, the combined ZnSO4 and Na2SeO3 treatment alleviated the inhibition of ZnSO4 and induced selenium content. Gene transcription and protein expression analyses revealed the changes in isothiocyanate and selenium metabolite levels in broccoli sprouts. ZnSO4 combined with Na2SeO3 was proven to activate a series of isothiocyanate metabolite genes (UGT74B1, OX1, and ST5b) and selenium metabolite genes (BoSultr1;1, BoCOQ5-2, and BoHMT1). The relative abundance of the total 317 and 203 proteins, respectively, in 4-day-old broccoli sprouts varied, and the metabolic and biosynthetic pathways for secondary metabolites were significantly enriched in ZnSO4/control and ZnSO4 combined Na2SeO3/ZnSO4 comparisons. The findings demonstrated how ZnSO4 combined with Na2SeO3 treatment reduced stress inhibition and the accumulation of encouraged selenium and isothiocyanates during the growth of broccoli sprouts.

Urinary excretion of organosulfur compounds after acute ingestion of black onion.

Onion (Allium cepa L.) and its newly derived product "black onion" are characterised by the presence of compounds with potential bioactivity, particularly organosulfur compounds (OSCs). However, little is known about the metabolism, distribution, and excretion of these compounds as they pass through the gastrointestinal tract. This study monitored healthy subjects after an acute intake of black onion and analysed the excretion of OSCs using UHPLC-HRMS. A total of 31 OSCs were detected in urine after the acute ingestion of black onion, the main components being S-methyl-L-cysteine sulfoxide (methiin) (13.6 ± 3.9 µmol), isoalliin (12.4 ± 4.7 µmol) and S-propyl-L-cysteine (deoxypropiin) (3.1 ± 0.7 µmol). Moreover, N-acetylated metabolites of the major OSCs detected in black onion, namely, N-acetyl-S-(1-propenyl)-L-cysteine sulfoxide (NAS1PCS) and N-acetyl-S-(1-propenyl)-L-cysteine (NAS1PC), were found in urine after black onion consumption. The N-acetylation reaction takes place in the kidneys and liver, and metabolism pathways are proposed to explain the excretion of OSCs in urine. The basis of the identification of OSCs as urinary metabolites after black onion consumption is described for the first time and provides the basis for further research.

Sadopeptins A and B, Sulfoxide- and Piperidone-Containing Cyclic Heptapeptides with Proteasome Inhibitory Activity from a Streptomyces sp.

New sulfur-bearing natural products, sadopeptins A and B (1 and 2), were discovered from Streptomyces sp. YNK18 based on a targeted search using the characteristic isotopic signature of sulfur in mass spectrometry analysis. Compounds 1 and 2 were determined to be new cyclic heptapeptides, bearing methionine sulfoxide [Met(O)] and 3-amino-6-hydroxy-2-piperidone (Ahp), based on 1D and 2D NMR spectroscopy along with IR, UV, and MS. The configurations of sadopeptins A and B (1 and 2) were established via the analysis of the ROESY NMR correlation, oxidation, Marfey's method, and circular dichroism (CD) spectroscopy. The bioinformatics analysis of the full Streptomyces sp. YNK18 genome identified a nonribosomal peptide synthetase (NRPS) biosynthetic gene cluster (BGC), and a putative biosynthetic pathway is proposed. Sadopeptins A and B displayed proteasome-inhibitory activity without affecting cellular autophagic flux.

Combined contributions of cytochrome P450s (CYPs) and non-enzymatic metabolism in the in vitro biotransformation of anaprazole, a novel proton pump inhibitor.

Anaprazole, a new proton pump inhibitor (PPI), is designed for the treatment of acid-related diseases, such as gastric ulcers and gastroesophageal reflux. This study explored the in vitro metabolic transformation of anaprazole. The metabolic stabilities of anaprazole in human plasma and human liver microsomes (HLM) were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Then, the contribution (%) of non-enzymatic and cytochrome P450s (CYPs) enzyme-mediated anaprazole metabolism was assessed. To obtain the metabolic pathways of anaprazole, the metabolites generated in HLM, thermal deactivated HLM, and cDNA-expressed recombinant CYPs incubation systems were identified by ultra-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (UPLC/Q-TOF-MS). Results showed that anaprazole was very stable in human plasma and unstable in HLM. The contribution (%) of non-enzymatic vs. CYPs enzyme-mediated metabolism was 49% vs. 51%. CYP3A4 was the major enzyme (48.3%), followed by CYP2C9 (17.7%) and CYP2C8 (12.3%), in responsible for the metabolism of anaprazole. Specific chemical inhibitors targeting CYP enzymes notably blocked the metabolic transformation of anaprazole. Six metabolites of anaprazole were identified in the non-enzymatic system, whereas 17 metabolites were generated in HLM. The biotransformation reactions mainly included sulfoxide reduction to thioether, sulfoxide oxidation to sulfone, deoxidation, dehydrogenation, O-dealkylation or O-demethylation of thioether, O-demethylation and dehydrogenation of thioether, O-dealkylation and dehydrogenation of thioether, thioether O-dealkylation and dehydrogenation of thioether, and O-dealkylation of sulfone. Both enzymatic and non-enzymatic metabolisms contribute to the clearance of anaprazole in human. Anaprazole is less likely to develop drug-drug interactions in clinical use compared to other PPIs.

Protective Effect of Sulforaphane on Oxidative Stress and Mitochondrial Dysfunction Associated with Status Epilepticus in Immature Rats.

The present study aimed to elucidate the effect of sulforaphane (a natural isothiocyanate) on oxidative stress and mitochondrial dysfunction during and at selected periods following status epilepticus (SE) induced in immature 12-day-old rats by Li-pilocarpine. Dihydroethidium was employed for the detection of superoxide anions, immunoblot analyses for 3-nitrotyrosine (3-NT) and 4-hydroxynonenal (4-HNE) levels and respiratory chain complex I activity for evaluation of mitochondrial function. Sulforaphane was given i.p. in two doses (5 mg/kg each), at PD 10 and PD 11, respectively. The findings of the present study indicate that both the acute phase of SE and the early period of epileptogenesis (1 week and 3 weeks following SE induction) are associated with oxidative stress (documented by the enhanced superoxide anion production and the increased levels of 3-NT and 4-HNE) and the persisting deficiency of complex I activity. Pretreatment with sulforaphane either completely prevented or significantly reduced markers of both oxidative stress and mitochondrial dysfunction. Since sulforaphane had no direct anti-seizure effect, the findings suggest that the ability of sulforaphane to activate Nrf2 is most likely responsible for the observed protective effect. Nrf2-ARE signaling pathway can be considered a promising target for novel therapies of epilepsy, particularly when new compounds, possessing inhibitory activity against protein-protein interaction between Nrf2 and its repressor protein Keap1, with less "off-target" effects and, importantly, with an optimal permeability and bioavailability properties, become available commercially.

Bioelectrochemical platform with human monooxygenases: FMO1 and CYP3A4 tandem reactions with phorate.

It is highly advantageous to devise an in vitro platform that can predict the complexity of an in vivo system. The first step of this process is the identification of a xenobiotic whose monooxygenation is carried out by two sequential enzymatic reactions. Pesticides are a good model for this type of tandem reactions since in specific cases they are initially metabolised by human flavin-containing monooxygenase 1 (hFMO1), followed by cytochrome P450 (CYP). To assess the feasibility of such an in vitro platform, hFMO1 is immobilised on glassy carbon electrodes modified with graphene oxide (GO) and cationic surfactant didecyldimethylammonium bromide (DDAB). UV-vis, contact angle and AFM measurements support the effective decoration of the GO sheets by DDAB which appear as 3 nm thick structures. hFMO1 activity on the bioelectrode versus three pesticides; fenthion, methiocarb and phorate, lead to the expected sulfoxide products with KM values of 29.5 ± 5.1, 38.4 ± 7.5, 29.6 ± 4.1 µM, respectively. Moreover, phorate is subsequently tested in a tandem system with hFMO1 and CYP3A4 resulting in both phorate sulfoxide as well as phoratoxon sulfoxide. The data demonstrate the feasibility of using bioelectrochemical platforms to mimic the complex metabolic reactions of xenobiotics within the human body.

Giardia duodenalis: Flavohemoglobin is involved in drug biotransformation and resistance to albendazole.

Giardia duodenalis causes giardiasis, a major diarrheal disease in humans worldwide whose treatment relies mainly on metronidazole (MTZ) and albendazole (ABZ). The emergence of ABZ resistance in this parasite has prompted studies to elucidate the molecular mechanisms underlying this phenomenon. G. duodenalis trophozoites convert ABZ into its sulfoxide (ABZSO) and sulfone (ABZSOO) forms, despite lacking canonical enzymes involved in these processes, such as cytochrome P450s (CYP450s) and flavin-containing monooxygenases (FMOs). This study aims to identify the enzyme responsible for ABZ metabolism and its role in ABZ resistance in G. duodenalis. We first determined that the iron-containing cofactor heme induces higher mRNA expression levels of flavohemoglobin (gFlHb) in Giardia trophozoites. Molecular docking analyses predict favorable interactions of gFlHb with ABZ, ABZSO and ABZSOO. Spectral analyses of recombinant gFlHb in the presence of ABZ, ABZSO and ABZSOO showed high affinities for each of these compounds with Kd values of 22.7, 19.1 and 23.8 nM respectively. ABZ and ABZSO enhanced gFlHb NADH oxidase activity (turnover number 14.5 min-1), whereas LC-MS/MS analyses of the reaction products showed that gFlHb slowly oxygenates ABZ into ABZSO at a much lower rate (turnover number 0.01 min-1). Further spectroscopic analyses showed that ABZ is indirectly oxidized to ABZSO by superoxide generated from the NADH oxidase activity of gFlHb. In a similar manner, the superoxide-generating enzyme xanthine oxidase was able to produce ABZSO in the presence of xanthine and ABZ. Interestingly, we find that gFlHb mRNA expression is lower in albendazole-resistant clones compared to those that are sensitive to this drug. Furthermore, all albendazole-resistant clones transfected to overexpress gFlHb displayed higher susceptibility to the drug than the parent clones. Collectively these findings indicate a role for gFlHb in ABZ conversion to its sulfoxide and that gFlHb down-regulation acts as a passive pharmacokinetic mechanism of resistance in this parasite.

Transcriptome analysis reveals the genes involved in S-alk(en)ylcysteine sulfoxide biosynthesis and its biosynthetic location in postharvest chive (Allium schoenoprasum L.).

The biosynthetic mechanism of S-alk(en)ylcysteine sulfoxides (CSOs), a flavor precursor and nonvolatile medicinal compound in chive is still poorly understood. In the present study, transcriptomic analysis was used to investigate the biosynthetic mechanism of S-alk(en)ylcysteine sulfoxides (CSOs) in green leaves of postharvest chive stored under normal temperature (20 °C) for 5 d and low-temperature (3 °C) for 12 d. The de novo assembly of the transcriptome enabled the identification of unigenes involved in the sulfur assimilation and CSOs biosynthesis. The RNA-seq data showed that the unigenes related to sulfur assimilation were down-regulated during storage under 20 °C and 3 °C. The low temperature did not affect cysteine biosynthesis and the expression of γ-glutamyl transpeptidase (GGT) and flavin-containing monooxygenase (FMO) involved in CSOs biosynthesis; nonetheless, it prolonged CSOs synthesis by sustaining the chive quality during the storage period. The qPCR data revealed that the expressions of genes related to sulfur assimilation were mainly in the white stalk. In contrast, CSOs biosynthetic genes had higher expression levels in green leaf. The results indicate the CSOs were mainly synthesized in green leaf while cysteine, the primary substrate for CSOs synthesis, was from de novo synthesis and proteolysis. The study presents discrete evidence that CSOs biosynthesis in postharvest chives occurs in green leaves and is translocated to the white stalk for storage.

Sulforaphane Acts Through NFE2L2 to Prevent Hypoxia-Induced Apoptosis in Porcine Granulosa Cells via Activating Antioxidant Defenses and Mitophagy.

In mammals, a vast majority of ovarian follicles undergo atresia, which is caused by granulosa cell (GC) apoptosis. GCs in follicles are exposed to low oxygen. Hypoxia triggers reactive oxygen species (ROS) generation, which leads to cell oxidative stress and apoptosis. Sulforaphane (SFN), a phytochemical isothiocyanate enriched in cruciferous vegetables, has exhibited a crucial role in mitigating oxidative stress. To explore the effect of SFN on porcine GC apoptosis in a hypoxic environment, we handled the established hypoxia model (1% O2) of cultured porcine GCs with SFN. Results showed that SFN rescued hypoxia-induced apoptosis and viability of GCs. Meanwhile, SFN increased the expression of antioxidant enzymes and reduced the accumulation of ROS in GC cytoplasm and mitochondria under hypoxia. Mechanically, SFN activated the transcription factor of redox-sensitive nuclear factor-erythroid 2-related factor 2 (NFE2L2) entering the nucleus, further inducing mitophagy and increased antioxidant capacity, finally alleviating the adverse effect of hypoxia on porcine GCs. In conclusion, SFN inhibited hypoxia-evoked GC apoptosis by activating antioxidant defenses and mitophagy through NFE2L2. New targets may be provided for regulating follicular development and atresia by these findings.

Daidzein-directed methionine γ-lyase in enzyme prodrug therapy against breast cancer.

Thiosulfinates in situ formed by "pharmacological pair" C115H methionine γ-lyase/S-(allyl/alkyl)-l-cysteine sulfoxides possess cytotoxic activity against various malignant cell lines. To investigate in vivo antitumor activity of thiosulfinates generated directly at the surface of tumor cells, a chemical conjugate between Clostridium novyi C115H methionine γ-lyase (C115H MGL) and isoflavone daidzein was prepared. The binding of conjugate (C115H-Dz) to various breast cancer cell lines was demonstrated, as well as its cytotoxicity in the presence of S-(allyl/alkyl)-l-cysteine sulfoxides. The most promising among thiosulfinates was dipropyl thiosulfinate (IC50 < 0.53 µM). The pharmacokinetic parameters of C115H MGL and C115H-Dz were obtained. Plasma half-lives of the enzyme and conjugated enzyme were 4.4 and 7.2 h, respectively. In vivo antitumor effect of pharmacological pairs on SKBR-3 xenografts was demonstrated. Treatment of tumor-bearing mice with a pair of C115H-Dz/propiin inhibited tumor growth by 85%.

Colorimetric High-Throughput Screening Method for Directed Evolution of Prazole Sulfide Monooxygenase.

Baeyer-Villiger monooxygenases (BVMOs) are important biocatalysts for the enzymatic synthesis of chiral sulfoxides, including chiral sulfoxide-type proton pump inhibitors for the treatment of gastrointestinal diseases. However, native BVMOs are not yet suitable for practical application due to their unsatisfactory activity and thermostability. Although protein engineering approaches can help address these issues, few feasible high-throughput methods are available for the engineering of such enzymes. Herein, a colorimetric detection method to distinguish sulfoxides from sulfides and sulfones was developed for prazole sulfide monooxygenases. Directed evolution enabled by this method has identified a prazole sulfide monooxygenase CbBVMO variant with improved activity and thermostability that catalyzes the asymmetric oxidation of lansoprazole sulfide. A 71.3 % increase in conversion and 6 °C enhancement in the melting point were achieved compared with the wild-type enzyme. This new method is feasible for high-throughput screening of prazole sulfide monooxygenase variants with improved activity, thermostability, and/or substrate specificity.

Formation of volatile sulfur compounds and S-methyl-l-cysteine sulfoxide in Brassica oleracea vegetables.

Besides glucosinolates, Brassica vegetables accumulate sulfur-containing (+)-S-methyl-l-cysteine sulfoxide (SMCSO, methiin), mainly known from Allium vegetables. Such (+)-S-alk(en)yl-l-cysteine sulfoxides can degrade to volatile organosulfur compounds (VOSCs), which have been linked to health beneficial effects. In the present study, the accumulation of SMCSO and the formation of VOSCs was investigated in Brassica oleracea vegetables. SMCSO content of commercially available white and red cabbages was monitored over a three-month period and linked with the formation of VOSCs. S-Methyl methanethiosulfinate was the main VOSC released from SMCSO. Upon heating, it degraded to dimethyltrisulfide and dimethyldisulfide, which were less abundant in fresh homogenates. SMCSO made up approximately 1% of the dry matter of cabbages and the overall contents were similar in white and red cabbages (3.2-10.2 and 3.9-10.3 µmol/g fresh weight, respectively). Using proteome profiling it was shown that recovery of VOSCs correlated with abundance of two isoforms of cystine lyase.

Influence of sequential exogenous pretreatment and contact ultrasound-assisted air drying on the metabolic pathway of glucoraphanin in broccoli florets.

In this investigation, the combinations of exogenous pretreatment (melatonin or vitamin C) and contact ultrasound-assisted air drying were utilized to dry broccoli florets. To understand the influences of the studied dehydration methods on the conversion of glucoraphanin to bioactive sulforaphane in broccoli, various components (like glucoraphanin, sulforaphane, myrosinase, etc.) and factors (temperature and moisture) involved in the metabolism pathway were analyzed. The results showed that compared with direct air drying, the sequential exogenous pretreatment and contact ultrasound drying shortened the drying time by 19.0-22.7%. Meanwhile, contact sonication could promote the degradation of glucoraphanin. Both melatonin pretreatment and vitamin C pretreatment showed protective effects on the sulforaphane content and myrosinase activity during the subsequent drying process. At the end of drying, the sulforaphane content in samples dehydrated by the sequential melatonin (or vitamin C) pretreatment and ultrasound-intensified drying was 14.4% (or 26.5%) higher than only air-dried samples. The correlation analysis revealed that the exogenous pretreatment or ultrasound could affect the enzymatic degradation of glucoraphanin and the generation of sulforaphane through weakening the connections of sulforaphane-myrosinase, sulforaphane-VC, and VC-myrosinase. Overall, the reported results can enrich the biochemistry knowledge about the transformation of glucoraphanin to sulforaphane in cruciferous vegetables during drying, and the combined VC/melatonin pretreatment and ultrasound drying is conducive to protect bioactive sulforaphane in dehydrated broccoli.