Rational Molecular Design of a Fluorescent Probe for Selectively Sensing Human Cytochrome P450 2D6.

Cytochrome P450 2D6 (CYP2D6) is a key enzyme that mediates the metabolism of various drugs and endogenous substances in humans. However, its biological role in drug-drug interactions especially mechanism-based inactivation (MBI), and various diseases remains poorly understood, owing to the lack of molecular tools suitable for selectively monitoring CYP2D6 in complex biological systems. Herein, using a tailored molecular strategy, we developed a fluorescent probe BDPM for CYP2D6. BDPM exhibits excellent specificity and imaging capability for CYP2D6, making it suitable for the real-time monitoring of endogenous CYP2D6 activity in living bio-samples. Therefore, our tailored strategy proved useful for constructing the highly selective and enzyme-activated fluorescent probes. BDPM as a molecular tool to explore the critical roles of CYP2D6 in the pathogenesis of diseases, high-throughput screening of inhibitors and intensive investigation of CYP2D6-induced MBI in natural systems.

Drug-drug interactions induced by Linderane based on mechanism-based inactivation of CYP2C9 and the molecular mechanisms.

Linderane (LDR) is a main furan-containing sesquiterpenoid of the common herbal medicine Lindera aggregata (Sims) Kosterm. Our early study indicated that LDR led to mechanism-based inactivation (MBI) of CYP2C9 in vitro, implying possible drug-drug interactions (DDIs) in clinic. In the present study, influence of LDR on the pharmacokinetics of the corresponding hydroxylated metabolites of CYP2C9 substrates in rats was investigated. Pharmacokinetic studies revealed that pretreatment with LDR at 20 mg/kg for 15 days inhibited the metabolism of both tolbutamide and warfarin catalyzed by CYP2C9. As for 4-hydroxytolbutamide, the Cmax was decreased, the t1/2z was prolonged, and the Vz/F was increased, all with significant difference. As for 7-hydroxywarfarin, the AUC0-t/AUC0-∞ and CLz/F were significantly decreased and increased, respectively. Furthermore, the underlying molecular mechanisms based on MBI of CYP2C9 by LDR were revealed. Two reactive metabolites of LDR, furanoepoxide and γ-ketoenal intermediates were identified in CYP2C9 recombinant enzyme incubation systems. Correspondingly, covalent modifications of lysine and cysteine residues of CYP2C9 protein were discovered in the CYP2C9 incubation system treated with LDR. The formation of protein adducts exhibited obvious time- and dose-dependence, which is consistent with the trend of enzyme inhibition caused by LDR in vitro. In addition to the apoprotein of CYP2C9, the heme content was significantly reduced after co-incubation with LDR. These data revealed that modification of both apoprotein and heme of CYP2C9 by reactive metabolites of LDR led to MBI of CYP2C9, therefore resulting in the inhibition of biotransformation of CYP2C9 substrates to their corresponding metabolites in vivo.

Rational Design of a Two-Photon Fluorescent Probe for Human Cytochrome†P450 3A and the Visualization of Mechanism-Based Inactivation.

Mechanism-based inactivation (MBI) can mediate adverse reactions and hepatotoxicity from drugs, which is a result of their conversion into highly reactive metabolites catalyzed by enzymes such as cytochrome P450 3A (CYP3A). In the present research, we optimized the key interaction domain of the fluorophore with the target protein to develop a two-photon fluorescent probe for CYP3A that is involved in the metabolism of more than half of all clinical drugs. The developed BN-1 probe exhibited appropriate selectivity and sensitivity for the semi-quantitative detection and imaging of endogenous CYP3A activity in various living systems, thereby providing a high-throughput screening system enabling evaluation of MBI-associated hepatotoxicity by CYP3A. Using BN-1 as a fluorescent molecular tool facilitates the efficient discovery and characterization of CYP3A-induced MBI in natural systems.

Mechanism-based inactivation of cytochrome P450 enzymes by natural products based on metabolic activation.

Cytochrome P450 enzymes (P450 enzymes) are the most common and important phase I metabolic enzymes and are responsible for the majority of the metabolism of clinical drugs and other xenobiotics. Drug-drug interactions (DDIs) can occur when the activities of P450 enzymes are inhibited. In particular, irreversible inhibition of P450 enzymes may lead to severe adverse interactions, compared to reversible inhibition. Many natural products have been shown to be irreversible inhibitors of P450 enzymes. The risks for intake of naturally occurring irreversible P450 enzyme inhibitors have been rising due to the rapid growth of the global consumption of natural products. Irreversible inhibition is usually called mechanism-based inactivation, which is time-, concentration- and NADPH- dependent. Generally, the formation of electrophilic intermediates is fundamental for the inactivation of P450 enzymes. This review comprehensively classifies natural P450 enzyme inactivators, including terpenoids, phenylpropanoids, flavonoids, alkaloids, and quinones obtained from herbs or foods. Moreover, the structure - activity correlations according to the IC50 (or Ki) values reported in the literature as well as the underlying mechanisms based on metabolic activation are highlighted in depth.

Engineering protonation conformation of l-aspartate-α-decarboxylase to relieve mechanism-based inactivation.

Mechanism-based inactivation of l-aspartate-α-decarboxylase (PanD), which leads to irreversible modification of active site, is a major challenge in the efficient production of ß-alanine from L-aspartic acid. In this study, a semi-rational strategy that combined conformational dynamics and structural alignment was applied to increase the catalytic stability of Bacillus subtilis PanD (BsPanD). Using site-saturation and C-terminal deletion, the variant Q5 (BsPanDI46V/I88M/K104S/I126* ) was generated. The catalytic half-life and the total turnover number (TTN) of Q5 were 3.48-fold and 2.52-fold higher, respectively, compared with that of the parent Q0. The reasons for the differences were the prolonged distance d1 between the phenolic group of Tyr58 and pyruvoyl group of Ser25 (4.9 Å in Q0 vs. 5.5 Å in Q5), an increased difficulty for incorrect protonation to occur, and the decreased flexibility of residues in regions A, B, and C, thereby enhancing the probability of correct protonation. Variant Q5, coupled with l-aspartase (AspA) in a 15-L bioreactor, generated a linear cascade system using fumaric acid as a substrate, yielding 118.6 g/L ß-alanine with a product/catalyst (P/C) ratio of 5.9 g/g and a conversion > 99%. These results showed that reshaping the protonation conformation of PanD can efficiently relieve mechanism-based inactivation and boost catalytic stability.

Mechanism-based inactivator of isocitrate lyases 1 and 2 from Mycobacterium tuberculosis.

Isocitrate lyase (ICL, types 1 and 2) is the first enzyme of the glyoxylate shunt, an essential pathway for Mycobacterium tuberculosis (Mtb) during the persistent phase of human TB infection. Here, we report 2-vinyl-d-isocitrate (2-VIC) as a mechanism-based inactivator of Mtb ICL1 and ICL2. The enzyme-catalyzed retro-aldol cleavage of 2-VIC unmasks a Michael substrate, 2-vinylglyoxylate, which then forms a slowly reversible, covalent adduct with the thiolate form of active-site Cys191 2-VIC displayed kinetic properties consistent with covalent, mechanism-based inactivation of ICL1 and ICL2 with high efficiency (partition ratio, <1). Analysis of a complex of ICL1:2-VIC by electrospray ionization mass spectrometry and X-ray crystallography confirmed the formation of the predicted covalent S-homopyruvoyl adduct of the active-site Cys191.

Mechanism-based inactivation of cytochrome P450 2D6 by chelidonine.

Chelidonine (CHE) is a major bioactive constituent of greater celandine, a plant used in traditional herbal medicines. CHE has widely been used as an analgesic in clinical settings. We evaluated the inhibitory effects of CHE on human cytochrome P450 enzymes. CHE produced time-, concentration-, and NADPH-dependent inhibition of CYP2D6, with K I and k inact values of 20.49 µM and 11.05 min -1 , respectively. Approximately 76% of CYP2D6 activity was suppressed after 9 minute incubation with CHE (50 µM). The loss of enzyme activity was not restored following dialysis. The estimated partition ratio of the inactivation was about 156. Quinidine, a competitive inhibitor of CYP2D6, attenuated the CHE-mediated enzyme inactivation, while glutathione and catalase/superoxide dismutase did not markedly ameliorate the inhibitory effect. Upon oxidation using potassium ferricyanide, the 15.1% activity of CYP2D6 was restored. These findings indicate that CHE acted as a mechanism-based inactivator of CYP2D6 and the observed effects may induce potential drug-drug interactions.

The potent mechanism-based inactivation of CYP2D6 and CYP3A4 with fusidic acid in in vivo bioaccumulation.

1. The accumulation of fusidic acid (FA) after multiple doses of FA has been reported on in previous studies but the related mechanisms have not been clarified fully. In the present study, we explain the mechanisms related to the mechanism-based inactivation of CYP2D6 and CYP3A4. 2. The irreversible inhibitory effects of FA on CYP2D6 and CYP3A4 were examined via a series of experiments, including: (a) time-, concentration- and NADPH-dependent inactivation, (b) substrate protection in enzyme inactivation and (c) partition ratio with recombinant human CYP enzymes. Metoprolol α-hydroxylation and midazolam 1'-hydroxylation were used as marker reactions for CYP2D6 and CYP3A4 activities, and HPLC-MS/MS measurement was also utilised. 3. FA caused to the time- and concentration-dependent inactivation of CYP2D6 and CYP3A4. About 55.8% of the activity of CYP2D6 and 75.8% of the activity of CYP3A4 were suppressed after incubation with 10 µM FA for 15 min. KI and kinact were found to be 2.87 µM and 0.033 min-1, respectively, for CYP2D6, while they were 1.95 µM and 0.029 min-1, respectively, for CYP3A4. Inhibition of CYP2D6 and CYP3A4 activity was found to require the presence of NADPH. Substrates of CYP2D6 and CYP3A4 showed that the enzymes were protected against the inactivation induced by FA. The estimated partition ratio for the inactivation was 7 for CYP2D6 and 12 for CYP3A4. 4. FA is a potent mechanism-based inhibitor of CYP2D6 and CYP3A4, which may explain the accumulation of FA in vivo.

Molecular engineering of L-aspartate-α-decarboxylase for improved activity and catalytic stability.

ß-Alanine is an important precursor for the production of food additives, pharmaceuticals, and nitrogen-containing chemicals. Compared with the conventional chemical routes for ß-alanine production, the biocatalytic routes using L-aspartate-α-decarboxylase (ADC) are more attractive when energy and environment are concerned. However, ADC's poorly understood properties and its inherent mechanism-based inactivation significantly limited the application of this enzyme. In this study, three genes encoding the ADC enzymes from Escherichia coli, Corynebacterium glutamicum, and Bacillus subtilis were overexpressed in E. coli. Their properties including specific activity, thermostability, and mechanism-based inactivation were characterized. The ADC enzyme from B. subtilis, which had higher specific activity and thermostability than the others, was selected for further study. In order to improve its activity and relieve its mechanism-based inactivation by molecular engineering so as to improve its catalytic stability, a high-throughput fluorometric assay of ß-alanine was developed. From a library of 4000 mutated enzymes, two variants with 18-22% higher specific activity and 29-64% higher catalytic stability were obtained. The best variant showed 50% higher ß-alanine production than the wild type after 8 h of conversion of L-aspartate, showing great potential for industrial biocatalytic production of ß-alanine.

Inactivation of the particulate methane monooxygenase (pMMO) in Methylococcus capsulatus (Bath) by acetylene.

Acetylene (HCCH) has a long history as a mechanism-based enzyme inhibitor and is considered an active-site probe of the particulate methane monooxygenase (pMMO). Here, we report how HCCH inactivates pMMO in Methylococcus capsulatus (Bath) by using high-resolution mass spectrometry and computational simulation. High-resolution MALDI-TOF MS of intact pMMO complexes has allowed us to confirm that the enzyme oxidizes HCCH to the ketene (C2H2O) intermediate, which then forms an acetylation adduct with the transmembrane PmoC subunit. LC-MS/MS analysis of the peptides derived from in-gel proteolytic digestion of the protein subunit identifies K196 of PmoC as the site of acetylation. No evidence is obtained for chemical modification of the PmoA or PmoB subunit. The inactivation of pMMO by a single adduct in the transmembrane PmoC domain is intriguing given the complexity of the structural fold of this large membrane-protein complex as well as the complicated roles played by the various metal cofactors in the enzyme catalysis. Computational studies suggest that the entry of hydrophobic substrates to, and migration of products from, the catalytic site of pMMO are controlled tightly within the transmembrane domain. Support of these conclusions is provided by parallel experiments with two related alkynes: propyne (CH3CCH) and trifluoropropyne (CF3CCH). Finally, we discuss the implication of these findings to the location of the catalytic site in pMMO.

Single concentration loss of activity assay provides an improved assessment of drug-drug interaction risk compared to IC50-shift.

1. The utility of two abbreviated, higher-throughput assays [IC50-shift and the loss of activity (LOA) assay] to evaluate time-dependent inhibition (TDI) of 24 structurally related compounds was compared. 2. Good correlation (R(2) = 0.90) between % inhibition and kinact/KI suggested that the LOA assay has utility as an indicator of TDI potential. Weaker correlation was observed for the shifted IC50 (IC50(T = 30)) (R(2) = 0.61) and the fold-shift in IC50 (R(2) = 0.17). 3. Primary mechanism for poor correlation was depletion of active enzyme at concentrations > 1 µM leading to greater than predicted inhibition in the IC50-shift assay. 4. Previously reported strong correlations between IC50(T = 30) and kinact/KI were found to be dependent on potent TDI compounds with kinact/KI > 30; correlation was reduced for moderate inhibitors (kinact/KI < 30). LOA assay maintained good correlation even when strong TDI compounds were excluded. 5. LOA assay (% Inhibition at 30 min, 10 µM) was a good predictor of in vivo DDI (AUCr), providing a graded response with low potential for false negatives or positives. IC50-shift assay had bias for over-predicting in vivo DDI and was more likely to identify false positives.

Mechanism-based inactivation of human cytochrome P450 1A2 and 3A4 isoenzymes by anti-tumor triazoloacridinone C-1305.

1. 5-Dimethylaminopropylamino-8-hydroxytriazoloacridinone, C-1305, is a promising anti-tumor therapeutic agent with high activity against several experimental tumors. 2. It was determined to be a potent and selective inhibitor of liver microsomal and human recombinant cytochrome P450 (CYP) 1A2 and 3A4 isoenzymes. Therefore, C-1305 might modulate the effectiveness of other drugs used in multidrug therapy. 3. The objective of this study was to investigate the mechanism of the observed C-1305-mediated inactivation of CYP1A2 and CYP3A4. 4. Our findings indicated that C-1305 produced a time- and concentration-dependent decrease in 7-ethoxycoumarin O-deethylation (CYP1A2, KI = 10.8 ± 2.14 µM) and testosterone 6ß-hydroxylation (CYP3A4, KI = 9.1 ± 2.82 µM). The inactivation required the presence of NADPH, was unaffected by a nucleophilic trapping agent (glutathione) and a reactive oxygen species scavenger (catalase), attenuated by a CYP-specific substrate (7-ethoxycoumarin or testosterone), and was not reversed by potassium ferricyanide. The estimated partition ratios of 1086 and 197 were calculated for the inactivation of CYP1A2 and CYP3A4, respectively. 5. In conclusion, C-1305 inhibited human recombinant CYP1A2 and CYP3A4 isoenzymes by mechanism-based inactivation. The obtained knowledge about specific interactions between C-1305 and/or its metabolites, and CYP isoforms would be useful for predicting the possible drug-drug interactions in potent multidrug therapy.

Mechanism-based inactivation of CYP450 enzymes: a case study of lapatinib.

Mechanism-based inactivation (MBI) of CYP450 enzymes is a unique form of inhibition in which the enzymatic machinery of the victim is responsible for generation of the reactive metabolite. This precondition sets up a time-dependency for the inactivation process, a hallmark feature that characterizes all MBI. Yet, MBI itself is a complex biochemical phenomenon that operates in different modes, namely, covalent binding to apoprotein, covalent binding of the porphyrin group and also complexation of the catalytic iron. Using lapatinib as a recent example of toxicological interest, we present an example of a mixed-function MBI that can confound clinical drug-drug interactions manifestation. Lapatinib exhibits both covalent binding to the apoprotein and formation of a metabolite-intermediate complex in an enzyme-selective manner (CYP3A4 versus CYP3A5), each with different reactive metabolites. The clinical implication of this effect is also contingent upon genetic polymorphisms of the enzyme involved as well as the co-administration of other substrates, inhibitors or inducers, culminating in drug-drug interactions. This understanding recapitulates the importance of applying isoform-specific mechanistic investigations to develop customized strategies to manage such outcomes.

Mechanism-based inactivation of CYP2C9 by linderane.

1. Linderane (LDR), a furan-containing sesquiterpenoid, is found in Lindera aggregata (Sims) Kosterm, a common traditional Chinese herbal medicine. We thoroughly studied the irreversible inhibitory effect of LDR on cytochrome P450 2C9 (CYP2C9). 2. LDR caused a time- and concentration-dependent inactivation of CYP2C9. In addition, the inactivation of CYP2C9 by LDR was NADPH-dependent and irreversible. More than 50% of CYP2C9 activity was lost after its incubation with LDR at the concentration of 10 µM for 15 min at 30 °C. The maximal rate constant for inactivation (kinact) was found to be 0.0419 min(-1), and the concentration required for half-maximal inactivation (KI) was 1.26 µM, respectively. Glutathione (GSH), catalase, and superoxide dismutase (SOD) failed to protect CYP2C9 against inactivation by LDR. Diclofenac, a substrate of CYP2C9, prevented the enzyme from inactivation produced by LDR. The estimated partition ratio of the inactivation was approximately 227. 3. Two reactive intermediates, including furanoepoxide and γ-ketoenal, might be responsible for the observed enzyme inactivation. The formation of the intermediates was verified by chemical synthesis. Multiple P450 enzymes, including CYPs 1A2, 2B6, 2C9, 2C19, 2D6, 3A4, and 3A5, were found to be involved in the metabolic activation of LDR. In conclusion, LDR was characterized as a mechanism-based inactivator of CYP2C9.

CYP2J2-mediated metabolism of arachidonic acid in heart: A review of its kinetics, inhibition and role in heart rhythm control.

Cytochrome P450 2 J2 (CYP2J2) is primarily expressed extrahepatically and is the predominant epoxygenase in human cardiac tissues. This highlights its key role in the metabolism of endogenous substrates. Significant scientific interest lies in cardiac CYP2J2 metabolism of arachidonic acid (AA), an omega-6 polyunsaturated fatty acid, to regioisomeric bioactive epoxyeicosatrienoic acid (EET) metabolites that show cardioprotective effects including regulation of cardiac electrophysiology. From an in vitro perspective, the accurate characterization of the kinetics of CYP2J2 metabolism of AA including its inhibition and inactivation by drugs could be useful in facilitating in vitro-in vivo extrapolations to predict drug-AA interactions in drug discovery and development. In this review, background information on the structure, regulation and expression of CYP2J2 in human heart is presented alongside AA and EETs as its endogenous substrate and metabolites. The in vitro and in vivo implications of the kinetics of this endogenous metabolic pathway as well as its perturbation via inhibition and inactivation by drugs are elaborated. Additionally, the role of CYP2J2-mediated metabolism of AA to EETs in cardiac electrophysiology will be expounded.

Time-dependent inhibition of CYP3A4 by sertraline, a selective serotonin reuptake inhibitor.

Drug-drug interactions associated with selective serotonin reuptake inhibitors (SSRIs) are widely known. A major interaction by SSRIs is the inhibition of cytochrome P450 (P450)-mediated hepatic drug metabolism. The SSRI, sertraline, is also reported to increase the blood concentration of co-administered drugs. The potency of sertraline directly to inhibit hepatic drug metabolism is relatively weak compared with the other SSRIs, implying that additional mechanisms are involved in the interactions. The study examined whether sertraline produces time-dependent inhibition of CYP3A4 and/or other P450 enzymes. Incubation of human liver microsomes with sertraline in the presence of NADPH resulted in marked decreases in testosterone 6ß-hydroxylation activities, indicating that sertraline metabolism leads to CYP3A4 inactivation. This inactivation required NADPH and was not protected by glutathione. No significant inactivation was observed for other P450 enzymes. Spectroscopic evaluation revealed that microsomes with and without sertraline in the presence of NADPH gave a Soret peak at 455 nm, suggesting the formation of metabolic intermediate (MI) complexes of sertraline metabolite(s) with the reduced form of P450. This is the first report indicating that sertraline produced time-dependent inhibition of CYP3A4, which may be associated with MI complex formation.

Diyne inactivators and activity-based fluorescent labeling of phenol hydroxylase in Pseudomonas sp. CF600.

An activity-based labeling (ABL) approach was investigated for the phenol-oxidizing bacterium, Pseudomonas sp. CF600. Phenol-grown cells were exposed to several different terminal diynes, and following cell breakage, extracts of these cells were added to copper-catalyzed alkyne/azide cycloaddition reactions containing Alexa Fluor 647 azide. Analysis of total cell proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and near-infrared scanning demonstrated covalent fluorescent labeling of a 58- and a 34-kDa polypeptide in all diyne-treated cell types. Further studies using 1,4-diethynylbenzene (DEB) demonstrated that these labeled polypeptides were consistently detected in cells grown on substrates that exhibited phenol-dependent O2 uptake activity but not observed when cells were grown on substrates such as dextrose or catechol that did not support this activity. Fluorescent labeling of the two polypeptides in DEB-treated, phenol-grown cells was time dependent and was inhibited by several known substrates for phenol hydroxylase. These results suggest that diverse diynes act as mechanism-based inactivators of phenol hydroxylase in Pseudomonas sp. CF600 and that this effect can be exploited by ABL approaches to selectively label the major 58- and 34-kDa subunits of the hydroxylase component of this complex enzyme.

Metabolic activation of drugs by cytochrome P450 enzymes: Biochemical insights into mechanism-based inactivation by fibroblast growth factor receptor inhibitors and chemical approaches to attenuate reactive metabolite formation.

Metabolic activation of drugs by cytochrome P450 enzymes (P450) to chemically reactive electrophiles is commonly regarded as a key molecular-initiating event underpinning idiosyncratic drug-induced liver injury. However, apart from precipitating toxicities, these labile intermediates can be sequestered within the P450 active site and engender a unique form of irreversible inhibition known as mechanism-based inactivation (MBI) which bears profound clinical implications (i.e., drug-drug interactions, autoinhibition of hepatic elimination, time-dependent and/or nonlinear pharmacokinetics). Consequently, there has been considerable attempts to develop medicinal chemistry strategies to attenuate or abolish metabolic activation and its deleterious downstream effects (i.e., MBI). In this review, we will first summarize the fundamental aspects and consequences of P450 metabolic activation with a focus on MBI. Following which, we will share our recent discoveries on the arcane metabolic activation pathways of an emerging class of tyrosine kinase inhibitors known as the fibroblast growth factor receptor (FGFR) inhibitors which in turn unravelled mechanistic insights into the biochemical basis and pharmacokinetic implications of its MBI. Finally, we will discuss, using relevant examples from the literature as well as from our laboratory, limitations of existing chemical approaches to minimize metabolic activation and highlight a promising new paradigm which involves the rational deuteration of a drug molecule at its known bioactivation 'hot-spot' to shunt metabolism away from these aberrant pathways and reduce reactive metabolite formation.

Rerouting Fluxes of the Central Carbon Metabolism and Relieving Mechanism-Based Inactivation of l-Aspartate-α-decarboxylase for Fermentative Production of ß-Alanine in Escherichia coli.

ß-Alanine, with the amino group at the ß-position, is an important platform chemical that has been widely applied in pharmaceuticals and feed and food additives. However, the current modest titer and productivity, increased fermentation cost, and complicated operation are the challenges for producing ß-alanine by microbial fermentation. In this study, a high-yield ß-alanine-producing strain was constructed by combining metabolic engineering, protein engineering, and fed-batch bioprocess optimization strategies. First, an aspartate-α-decarboxylase from Bacillus subtilis was introduced in Escherichia coli W3110 to construct an initial ß-alanine-producing strain. Production of ß-alanine was obviously increased to 4.36 g/L via improving the metabolic flux and reducing carbon loss by rerouting fluxes of the central carbon metabolism. To further increase ß-alanine production, mechanism-based inactivation of aspartate-α-decarboxylase was relieved by rational design to maintain the productivity at a high level in ß-alanine fed-batch fermentation. Finally, fed-batch bioprocess optimization strategies were used to improve ß-alanine production to 85.18 g/L with 0.24 g/g glucose yield and 1.05 g/L/h productivity in fed-batch fermentation. These strategies can be effectively used in the construction of engineered strains for ß-alanine and production of its derivatives, and the final engineered strain was a valuable microbial cell factory that can be used for the industrial production of ß-alanine.

Human cytochrome P450 3A-mediated two-step oxidation metabolism of dimethomorph: Implications in the mechanism-based enzyme inactivation.

Dimethomorph (DMM), an effective and broad-spectrum fungicide applied in agriculture, is toxic to environments and living organisms due to the hazardous nature of its toxic residues. This study aims to investigate the human cytochrome P450 enzyme (CYP)-mediated oxidative metabolism of DMM by combining experimental and computational approaches. Dimethomorph was metabolized predominantly through a two-step oxidation process mediated by CYPs, and CYP3A was identified as the major contributor to DMM sequential oxidative metabolism. Meanwhile, DMM elicited the mechanism-based inactivation (MBI) of CYP3A in a suicide manner, and the iminium ion and epoxide reactive intermediates generated in DMM metabolism were identified as the culprits of MBI. Furthermore, three common pesticides, prochloraz (PCZ), difenoconazole (DFZ) and chlorothalonil (CTL), could significantly inhibit CYP3A-mediated DMM metabolism, and consequently trigger elevated exposure to DMM in vivo. Computational studies elucidated that the differentiation effects in charge distribution and the interaction pattern played crucial roles in DMM-induced MBI of CYP3A4 during sequential oxidative metabolism. Collectively, this study provided a global view of the two-step metabolic activation process of DMM mediated by CYP3A, which was beneficial for elucidating the environmental fate and toxicological mechanism of DMM in humans from a new perspective.