FAD-containing Monooxygenase Research Articles

Bioorganometallic Chemistry at the Interface with Biocatalysis: Chemoselective Reduction of Biomimetic NAD+ Cofactors with [Cp*Rh(bpy)H]+, Tandem Catalysis with 1,4-NADH-Dependent Enzymes, Chiral Synthesis, Organotin Metabolites, and DFT Mechanism Studies

This Review will focus on our studies of the utilization of the [Cp*Rh(bpy)H]+ complex as a biomimetic hydrogenase, regeneration reagent for chemoselective reduction of biomimetic NAD+ cofactors, N-benzylnicotinamide triflate, 1, β-nicotinamide ribose-5′-methyl phosphate, 2, and natural NAD+, 3, in the conversion to their 1,4-NADH analogues, 1,4-dihydro-N-benzylnicotinamide, 4, and β-1,4-dihydronicotinamide-5′-ribose methyl phosphate, 5, and natural 1,4-NADH, 6, respectively. This regeneration reaction with 1,4-NADH biomimetics 4 and 5 was used in tandem with 1,4-NADH-dependent enzymes for organic synthesis. The enzyme examples will encompass horse liver alcohol dehydrogenase (HLADH) reductions of prochiral ketones to chiral S-alcohols; cytochrome P450 enzymes for selective C–H oxidation reactions of organic compounds, and organometallic substrates, tributyltin acetate and cyclohexyltriphenyltin, with 1,4-NAD(P)H; and the biomimetic 1,4-NADH cofactor, 4, with the 2-hydroxybiphenyl-3-monooxygenase enzyme, HbpA, a FAD-containing monooxygenase enzyme, for hydroxylation of ortho-substituted phenols to catechols. This Review also includes discussions on in-depth DFT mechanism studies, and other chemical mechanistic aspects, of all tandem catalytic reactions that demonstrate, utilizing a simplified model of the HLADH-Zn(II)-mediated enzyme, the unprecedented role of Zn(II) in the hydride transfer reaction. Moreover, site-directed mutagenesis with 1,4-NAD(P)H-dependent cytochrome P450 BM-3 improved the molecular recognition process for biomimetic 1,4-NADH cofactors in organic compound C–H oxidation reactions. The 1,4-NADH-dependent cytochrome P450 CAM recognized the biomimetic 1,4-NADH cofactors; however, site-directed mutagenesis of the putidaredoxin reductase (PdR) component increased the kinetics for the C–H oxidation reaction of camphor. Cyclohexyltriphenyltin demonstrated the differences in hydroxylation regioselectivity, when comparing the cytochrome P450 enzyme with biomimetic P450 porphyrin Fe(III) and Mn(III) models. The biomimetic 1,4-NADH cofactor, 1,4-dihydro-N-benzylnicotinamide, 4, was also utilized as a reducing agent, providing the reduced, chemoselective coenzyme, the FADH– anion, that further reacts with O2/H+ to provide the 4α-hydroperoxide flavin, for the HbpA enzymatic oxidative conversion of ortho-substituted phenols to catechols.

Transcriptional regulation of secondary metabolism and autophagy genes in response to DNA replication stress in Setosphaeria turcica

The fungal pathogen Setosphaeria turcica causes northern corn leaf blight (NCLB), which leads to considerable crop losses. Setosphaeria turcica elaborates a specialized infection structures called appressorium for maize infection. Previously, we demonstrated that the S. turcica triggers an S-phase checkpoint and ATR (Ataxia Telangiectasia and Rad3 related)-dependent self-protective response to DNA genotoxic insults during maize infection. However, how the regulatory mechanism works was still largely unknown. Here, we report a genome wide transcriptional profile analysis during appressorium formation in the present of DNA replication stress. We performed RNA-Seq analysis to identify S. tuicica genes responsive to DNA replication stress. In the current work, we found that appressorium-mediated maize infection by S. turcica is significantly blocked by S-phase checkpoint. A large serial of secondary metabolite and melanin biosynthesis genes were blocked in appressorium formation of S. turcica during the replication stress. The secondary metabolite biosynthesis genes including alcohol dehydrogenase GroES-like domain, multicopper oxidase, ABC-transporter families, cytochrome P450 and FAD-containing monooxygenase were related to plant pathogen infection. In addition, we demonstrated that autophagy in S. turcica is up-regulated by ATR as a defense response to stress. We identified StATG3, StATG4, StATG5, StATG7 and StATG16 genes for autophagy were induced by ATR-mediated S-phase checkpoint. We therefore propose that in response to genotoxic stress, S. turcica utilizes ATR-dependent pathway to turn off transcription of genes governing appressorium-mediated infection, and meanwhile inducing transcription of autophagy genes likely as a mechanism of self-protection, aside from the more conservative responses in eukaryotes.

Substrate binding tunes the reactivity of hispidin 3-hydroxylase, a flavoprotein monooxygenase involved in fungal bioluminescence

Fungal bioluminescence was recently shown to depend on a unique oxygen-dependent system of several enzymes. However, the identities of the enzymes did not reveal the full biochemical details of this process, as the enzymes do not bear resemblance to those of other luminescence systems, and thus the properties of the enzymes involved in this fascinating process are still unknown. Here, we describe the characterization of the penultimate enzyme in the pathway, hispidin 3-hydroxylase, from the luminescent fungus Mycena chlorophos (McH3H), which catalyzes the conversion of hispidin to 3-hydroxyhispidin. 3-Hydroxyhispidin acts as a luciferin substrate in luminescent fungi. McH3H was heterologously expressed in Escherichia coli and purified by affinity chromatography with a yield of 100 mg/liter. McH3H was found to be a single component monomeric NAD(P)H-dependent FAD-containing monooxygenase having a preference for NADPH. Through site-directed mutagenesis, based on a modeled structure, mutant enzymes were created that are more efficient with NADH. Except for identifying the residues that tune cofactor specificity, these engineered variants may also help in developing new hispidin-based bioluminescence applications. We confirmed that addition of hispidin to McH3H led to the formation of 3-hydroxyhispidin as sole aromatic product. Rapid kinetic analysis revealed that reduction of the flavin cofactor by NADPH is boosted by hispidin binding by nearly 100-fold. Similar to other class A flavoprotein hydroxylases, McH3H did not form a stable hydroperoxyflavin intermediate. These data suggest a mechanism by which the hydroxylase is tuned for converting hispidin into the fungal luciferin.

Monitoring the Reductive and Oxidative Half-Reactions of a Flavin-Dependent Monooxygenase using Stopped-Flow Spectrophotometry

Aspergillus fumigatus siderophore A (SidA) is an FAD-containing monooxygenase that catalyzes the hydroxylation of ornithine in the biosynthesis of hydroxamate siderophores that are essential for virulence (e.g. ferricrocin or N',N",N'''-triacetylfusarinine C)1. The reaction catalyzed by SidA can be divided into reductive and oxidative half-reactions (Scheme 1). In the reductive half-reaction, the oxidized FAD bound to Af SidA, is reduced by NADPH2,3. In the oxidative half-reaction, the reduced cofactor reacts with molecular oxygen to form a C4a-hydroperoxyflavin intermediate, which transfers an oxygen atom to ornithine. Here, we describe a procedure to measure the rates and detect the different spectral forms of SidA using a stopped-flow instrument installed in an anaerobic glove box. In the stopped-flow instrument, small volumes of reactants are rapidly mixed, and after the flow is stopped by the stop syringe (Figure 1), the spectral changes of the solution placed in the observation cell are recorded over time. In the first part of the experiment, we show how we can use the stopped-flow instrument in single mode, where the anaerobic reduction of the flavin in Af SidA by NADPH is directly measured. We then use double mixing settings where Af SidA is first anaerobically reduced by NADPH for a designated period of time in an aging loop, and then reacted with molecular oxygen in the observation cell (Figure 1). In order to perform this experiment, anaerobic buffers are necessary because when only the reductive half-reaction is monitored, any oxygen in the solutions will react with the reduced flavin cofactor and form a C4a-hydroperoxyflavin intermediate that will ultimately decay back into the oxidized flavin. This would not allow the user to accurately measure rates of reduction since there would be complete turnover of the enzyme. When the oxidative half-reaction is being studied the enzyme must be reduced in the absence of oxygen so that just the steps between reduction and oxidation are observed. One of the buffers used in this experiment is oxygen saturated so that we can study the oxidative half-reaction at higher concentrations of oxygen. These are often the procedures carried out when studying either the reductive or oxidative half-reactions with flavin-containing monooxygenases. The time scale of the pre-steady-state experiments performed with the stopped-flow is milliseconds to seconds, which allow the determination of intrinsic rate constants and the detection and identification of intermediates in the reaction4. The procedures described here can be applied to other flavin-dependent monooxygenases.5,6

Monitoring the reductive and oxidative half-reactions of a flavin-dependent monooxygenase using stopped-flow spectrophotometry.

Aspergillus fumigatus siderophore A (SidA) is an FAD-containing monooxygenase that catalyzes the hydroxylation of ornithine in the biosynthesis of hydroxamate siderophores that are essential for virulence (e.g. ferricrocin or N',N",N'''-triacetylfusarinine C)1. The reaction catalyzed by SidA can be divided into reductive and oxidative half-reactions (Scheme 1). In the reductive half-reaction, the oxidized FAD bound to Af SidA, is reduced by NADPH2,3. In the oxidative half-reaction, the reduced cofactor reacts with molecular oxygen to form a C4a-hydroperoxyflavin intermediate, which transfers an oxygen atom to ornithine. Here, we describe a procedure to measure the rates and detect the different spectral forms of SidA using a stopped-flow instrument installed in an anaerobic glove box. In the stopped-flow instrument, small volumes of reactants are rapidly mixed, and after the flow is stopped by the stop syringe (Figure 1), the spectral changes of the solution placed in the observation cell are recorded over time. In the first part of the experiment, we show how we can use the stopped-flow instrument in single mode, where the anaerobic reduction of the flavin in Af SidA by NADPH is directly measured. We then use double mixing settings where Af SidA is first anaerobically reduced by NADPH for a designated period of time in an aging loop, and then reacted with molecular oxygen in the observation cell (Figure 1). In order to perform this experiment, anaerobic buffers are necessary because when only the reductive half-reaction is monitored, any oxygen in the solutions will react with the reduced flavin cofactor and form a C4a-hydroperoxyflavin intermediate that will ultimately decay back into the oxidized flavin. This would not allow the user to accurately measure rates of reduction since there would be complete turnover of the enzyme. When the oxidative half-reaction is being studied the enzyme must be reduced in the absence of oxygen so that just the steps between reduction and oxidation are observed. One of the buffers used in this experiment is oxygen saturated so that we can study the oxidative half-reaction at higher concentrations of oxygen. These are often the procedures carried out when studying either the reductive or oxidative half-reactions with flavin-containing monooxygenases. The time scale of the pre-steady-state experiments performed with the stopped-flow is milliseconds to seconds, which allow the determination of intrinsic rate constants and the detection and identification of intermediates in the reaction4. The procedures described here can be applied to other flavin-dependent monooxygenases.5,6

A Systematic and Comprehensive Combinatorial Approach to Simultaneously Improve the Activity, Reaction Specificity, and Thermal Stability of p-Hydroxybenzoate Hydroxylase

We have simultaneously improved the activity, reaction specificity, and thermal stability of p-hydroxybenzoate hydroxylase by means of systematic and comprehensive combinatorial mutagenesis starting from available single mutations. Introduction of random mutations at the positions of four cysteine and eight methionine residues provided 216 single mutants as stably expressed forms in Escherichia coli host cells. Four characteristics, hydroxylase activity toward p-hydroxybenzoate (main activity), protocatechuate-dependent NADPH oxidase activity (sub-activity), ratio of sub-activity to main activity (reaction specificity), and thermal stability, of the purified mutants were determined. To improve the above characteristics for diagnostic use of the enzyme, 11 single mutations (C152V, C211I, C332A, M52V, M52Q, M110L, M110I, M213G, M213L, M276Q, and M349A) were selected for further combinatorial mutagenesis. All possible combinations of the mutations provided 18 variants with double mutations and further combinatorial mutagenesis provided 6 variants with triple mutations and 9 variants with quadruple mutations with the simultaneously improved four properties.

Metabolism and Disposition of a Selective α7 Nicotinic Acetylcholine Receptor Agonist in Humans

The metabolism and disposition of N-(3R)-1-azabicyclo[2.2.2]oct-3-ylfuro[2,3-c]pyridine-5-carboxamide (1), an alpha(7) nicotinic acetylcholinergic receptor agonist, were elucidated in humans (4 female, 4 male; all white) after an oral dose of [(3)H]1. Overall, 1 was well tolerated, with >94% of administered radioactivity excreted renally by 48 h postdose; lyophilization of all urine and plasma samples confirmed (3)H stability within [(3)H]1. Across genders, 1 underwent low-to-moderate oral clearance comprising both renal (67%) and metabolic (33%) components, with the biotransformation of 1 occurring predominantly via oxidation of its furanopyridine moiety to carboxylic acid 2, and minimally by modification of its quinuclidine nitrogen to N-oxide 4 or N-glucuronide M5. Experiments using human in vitro systems were undertaken to better understand the enzyme(s) involved in the phase 1 biotransformation pathways. The formation of 2 was found to be mediated by CYP2D6, a polymorphically expressed enzyme absent in 5 to 10% of white people, whereas the generation of 4 was catalyzed by CYP2D6, FAD-containing monooxygenase 1 (FMO1), and FMO3. It is of interest that, although no overall gender-related differences in excretory routes, mass recoveries, pharmacokinetics, or metabolite profiles of 1 were evident, the observation of one of eight subjects (13%) showing disparate (relative to all other volunteers) systemic exposures to 1, and urinary and plasma quantitative profiles nearly devoid of 2 with the highest levels of 1, seem consistent with both the identification of CYP2D6 as the only major recombinant cytochrome P450 transforming 1 to 2 and the demographics of white CYP2D6 poor metabolizers. Data also reported herein suggest that 4 is generated predominantly by renal FMO1 in humans.

Metabolic Activation of Thiacetazone by the Flavin Monooxygenase EtaA of Mycobacterium tuberculosis

Thiacetazone (TAZ) and ethionamide (ETA) are thioamide-containing second line antitubercular prodrugs for which there is an impressive clinical history of cross-resistance in M. tuberculosis and M. leprae. The recently identified FAD-containing monooxygenase EtaA has previously been shown to oxidize ETA to a sulfenic acid (-SOH) and eventually to the amide. EtaA is shown here to also be responsible for activation of TAZ. The two metabolites identified in the in vitro oxidation of TAZ by heterologously expressed EtaA are a sulfinic acid (-SO2H) and a carbodiimide (-N=C=NH), both of which apparently derive from an initially formed but not detected sulfenic acid intermediate. The pH dependence of the oxidation of TAZ by EtaA demonstrated that under basic conditions formation of the carbodiimide metabolite is favored, whereas under neutral or acidic conditions the sulfinic acid is preferred. The sulfenic acid intermediate undergoes redox cycling with glutathione whereas the carbodiimide metabolite forms a covalent adduct with glutathione. Analogous reactions are observed with the human flavin monooxygenases FMO1 and FMO2. The metabolites formed provide a possible rationale for the cytotoxicity of TAZ. This work was supported by NIH grant GM56531.

Discovery of a thermostable Baeyer–Villiger monooxygenase by genome mining

Baeyer-Villiger monooxygenases represent useful biocatalytic tools, as they can catalyze reactions which are difficult to achieve using chemical means. However, only a limited number of these atypical monooxygenases are available in recombinant form. Using a recently described protein sequence motif, a putative Baeyer-Villiger monooxygenase (BVMO) was identified in the genome of the thermophilic actinomycete Thermobifida fusca. Heterologous expression of the respective protein in Escherichia coli and subsequent enzyme characterization showed that it indeed represents a BVMO. The NADPH-dependent and FAD-containing monooxygenase is active with a wide range of aromatic ketones, while aliphatic substrates are also converted. The best substrate discovered so far is phenylacetone (k(cat) = 1.9 s(-1), K(M) = 59 microM). The enzyme exhibits moderate enantioselectivity with alpha-methylphenylacetone (enantiomeric ratio of 7). In addition to Baeyer-Villiger reactions, the enzyme is able to perform sulfur oxidations. Different from all known BVMOs, this newly identified biocatalyst is relatively thermostable, displaying an activity half-life of 1 day at 52 degrees C. This study demonstrates that, using effective annotation tools, genomes can efficiently be exploited as a source of novel BVMOs.

The Prodrug Activator EtaA from Mycobacterium tuberculosis Is a Baeyer-Villiger Monooxygenase

EtaA is a newly identified FAD-containing monooxygenase that is responsible for activation of several thioamide prodrugs in Mycobacterium tuberculosis. It was found that purified EtaA displays a remarkably low activity with the antitubercular prodrug ethionamide. Hinted by the presence of a Baeyer-Villiger monooxygenase sequence motif in the EtaA sequence, we have been able to identify a large number of novel EtaA substrates. It was discovered that the enzyme converts a wide range of ketones to the corresponding esters or lactones via a Baeyer-Villiger reaction, indicating that EtaA represents a Baeyer-Villiger monooxygenase. With the exception of aromatic ketones (phenylacetone and benzylacetone), long-chain ketones (e.g. 2-hexanone and 2-dodecanone) also are converted. EtaA is also able to catalyze enantioselective sulfoxidation of methyl-p-tolylsulfide. Conversion of all of the identified substrates is relatively slow with typical k(cat) values of around 0.02 s(-1). The best substrate identified so far is phenylacetone (K(m) = 61 microM, k(cat) = 0.017 s(-1)). Redox monitoring of the flavin cofactor during turnover of phenylacetone indicates that a step in the reductive half-reaction is limiting the rate of catalysis. Intriguingly, EtaA activity could be increased by one order of magnitude by adding bovine serum albumin. This reactivity and substrate acceptance-profiling study provides valuable information concerning this newly identified prodrug activator from M. tuberculosis.

Fermentation of 4-aminobutyrate by Clostridium aminobutyricum: cloning of two genes involved in the formation and dehydration of 4-hydroxybutyryl-CoA.

Clostridium aminobutyricum ferments 4-aminobutyrate via succinic semialdehyde, 4-hydroxybutyrate, 4-hydroxybutyryl-CoA and crotonyl-CoA to acetate and butyrate. The genes coding for the enzymes that catalyse the interconversion of these intermediates are arranged in the order abfD (4-hydroxybutyryl-CoA dehydratase), abfT (4-hydroxybutyrate CoA-transferase), and abfH (NAD-dependent 4-hydroxybutyrate dehydrogenase). The genes abfD and abfT were cloned, sequenced and expressed as active enzymes in Escherichia coli. Hence the insertion of the [4Fe-4S]clusters and FAD into the dehydratase required no additional specific protein from C. aminobutyricum. The amino acid sequences of the dehydratase and the CoA-transferase revealed close relationships to proteins deduced from the genomes of Clostridium difficile, Porphyromonas gingivalis and Archaeoglobus fulgidus. In addition the N-terminal part of the dehydratase is related to those of a family of FAD-containing mono-oxygenases from bacteria. The putative assignment in the databank of Cat2 (OrfZ) from Clostridium kluyveri as 4-hydroxybutyrate CoA-transferase, which is thought to be involved in the reductive pathway from succinate to butyrate, was confirmed by sequence comparison with AbfT (57% identity). Furthermore, an acetyl-CoA:4-hydroxybutyrate CoA-transferase activity could be detected in cell-free extracts of C. kluyveri. In contrast to glutaconate CoA-transferase from Acidaminococcus fermentans, mutation studies suggested that the glutamate residue of the motive EXG, which is conserved in many homologues of AbfT, does not form a CoA-ester during catalysis.

Role of ethanol exposure on cocaine metabolism in rat hepatocytes.

Cocaine remains a widely abused illicit substance in our society. Cocaine hepatotoxicity has been linked to cocaine metabolism. Cocaine can undergo hydrolytic inactivation via plasma and hepatic esterases or it can be N-oxidized by cytochrome P-450 and FAD-containing monooxygenases. Ethanol is frequently used in combination with cocaine. The presence of ethanol can affect the metabolism of other agents, depending on the dose and duration of exposure. In this investigation, hepatocytes isolated from male Sprague-Dawley rats were utilized to study the effect of ethanol exposure on cocaine metabolism. Hepatocytes were isolated using a two-step collagenase perfusion system. Hepatocytes (2 x 10(6) cells ml(-1)) were exposed to cocaine, ethanol or the combination of cocaine and ethanol for a 2-h period in a shaking water-bath at 30 oscillations per minute maintained at 37 degrees C. Sodium fluoride (NaF) was added to aliquots of cells which were removed from the incubation following 30, 60 and 120 min. The cells were homogenized on ice and immediately extracted for the quantification of cocaine, benzoylecognine, norcocaine and ethylcocaine by HPLC. Quantitative analysis revealed that there was a time-dependent increase in the disappearance of cocaine from hepatocytes. The rate of cocaine disappearance was not changed when ethanol was included in incubations containing cocaine. However, in the presence of ethanol there was a difference in the quantities of cocaine metabolites produced. When ethanol was included in incubations containing cocaine, the formation of norcocaine and benzoylecognine was less than that formed in hepatocytes exposed to cocaine alone. Additionally, when hepatocytes were exposed to cocaine in combination with ethanol, the formation of ethylcocaine was linear with time. This study revealed that in the presence of ethanol, cocaine qualitative metabolism is altered.

Structure and mutational analysis of Rab GDP-dissociation inhibitor.

The crystal structure of the bovine alpha-isoform of Rab GDP-dissociation inhibitor (GDI), which functions in vesicle-membrane transport to recycle and regulate Rab GTPases, has been determined to a resolution of 1.81 A. GDI is constructed of two main structural units, a large complex multisheet domain I and a smaller alpha-helical domain II. The structural organization of domain I is surprisingly closely related to FAD-containing monooxygenases and oxidases. Sequence-conserved regions common to GDI and the choroideraemia gene product, which delivers Rab to catalytic subunits of Rab geranylgeranyltransferase II, are clustered on one face of the molecule. The two most sequence-conserved regions, which form a compact structure at the apex of GDI, are shown by site-directed mutagenesis to play a critical role in the binding of Rab proteins.

An essential lysyl residue (Lys208) in the substrate-binding site of porcine FAD-containing monooxygenase.

The substrate (amine)-binding site of porcine FAD-containing monooxygenase (FMO) (EC 1.14.13.8) was examined using pyridoxal 5'-phosphate (pyridoxal-P) to modify lysyl residues. The enzymic activity of the FMO was inhibited competitively by pyridoxal-P. Upon reduction of pyridoxal-P-treated FMO with NaBH4, a new characteristic absorption peak of substituted pyridoxal-P appeared at 325 nm. The amino acid residue compositions of the native and pyridoxal-P-treated FMOs indicated that the lysyl residues were modified by pyridoxal-P. The about 74% inactivation of the enzymic activity on covalent pyridoxal-P treatment of the FMO was nearly completely prevented in the presence of the substrate, N,N-dimethylaniline. The FMO covalently modified with pyridoxal-P in the presence or absence of N,N-dimethylaniline was digested with trypsin treated with tosylphenylalanylchloromethane and the resultant peptide fragments were separated with a reverse-phase high-performance liquid chromatography system; only one peptide was specifically labeled with pyridoxal-P and was detected at 325 nm in the absence of N,N-dimethylaniline. The modified peptide was analyzed and identified as that comprising the amino acid residues 186-208. These results suggest that Lys208 plays an important role in the substrate (amine)-binding site of FMO.

An Essential Lysyl Residue (Lys208) in the Substrate‐Binding Site of Porcine FAD‐Containing Monooxygenase

The substrate (amine)‐binding site of porcine FAD‐containing monooxygenase (FMO) (EC 1.14.13.8) was examined using pyridoxal 5′‐phosphate (pyridoxal‐P) to modify lysyl residues. The enzymic activity of the FMO was inhibited competitively by pyridoxal‐P. Upon reduction of pyridoxal‐P ‐treated FMO with NaBH4, a new characteristic absorption peak of substituted pyridoxal‐P appeared at 325 nm. The amino acid residue compositions of the native and pyridoxal‐P ‐treated FMOs indicated that the lysyl residues were modified by pyridoxal‐P. The about 74% inactivation of the enzymic activity on covalent pyridoxal‐P treatment of the FMO was nearly completely prevented in the presence of the substrate, N,N ‐dimethylaniline. The FMO covalently modified with pyridoxal‐P in the presence or absence of N,N ‐dimethylaniline was digested with trypsin treated with tosylphenylalanylchloromethane and the resultant peptide fragments were separated with a reverse‐phase high‐performance liquid chromatography system; only one peptide was specifically labeled with pyridoxal‐P and was detected at 325 nm in the absence of N,N ‐dimethylaniline. The modified peptide was analyzed and identified as that comprising the amino acid residues 186–208.These results suggest that Lys208 plays an important role in the substrate (amine)‐binding site of FMO.

Inhibition of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine metabolic activity of porcine FAD-containing monooxygenase by selective monoamine oxidase-B inhibitors

The MPTP metabolic activity of porcine FAD-containing monooxygenase (FMO) (EC 1.14.13.8) was inhibited considerably by deprenyl and pargyline, selective MAO-B inhibitors, and they showed typical competitive inhibition. Deprenyl and pargyline, amine derivatives were also examined as to whether they are substrates for the FMO. It was found that deprenyl and pargyline are excellent substrates for the FMO. The K i and K m values of deprenyl and pargyline for the FMO are 14 μM and 9 μM, and 14.3 μM and 11.6 μM, at pH 8.0 and 25°C, respectively.

Characteristic properties and kinetic analysis with neurotoxins of porcine FAD-containing monooxygenase

An FAD-containing monooxygenase (EC 1.14.13.8) was purified from porcine liver microsomes by a new purification procedure and confirmed to give an electrophoretically single protein band. The optical and CD spectra, fluorescence and molar extinction coefficients of the FMO were investigated. The activity of the FMO was examined kinetically with neurotoxins, 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP), 1,2,3,4-tetrahydroisoquinoline (TIQ), and 1-methyl-6,7-dihydroxytetrahydroisoquinoline (MDTIQ), as substrates. The kinetic parameters of the FMO for the neurotoxins, molecular oxygen and electron donors were determined, in comparison, on with those of dimethylaniline. The CD spectrum of the FMO was measured in the absence and presence of NADP +, dimethylaniline or both. The results showed that the FMO metabolized the neurotoxins, and that NADH was a weak electron donor for it. The CD spectrum of the FMO in the oxidized form, which acts as an oxidase and oxygenase, unlike that of d-amino-acid oxidase, showed negative ellipticity, the secondary structure of the FMO changed, and the α-helix structure of the monooxygenase was affected by the formation of a complex of the FMO with NADP +, DMA or both.

Trimethylamine N-oxygenation and N-demethylation in rat liver microsomes

The in vitro oxidation of trimethylamine (TMA) to TMA N-oiade (TMAO) and dimethyl-amine (DMA) was studied in rat liver microsomes. Pretreatment of rats with phenobarbital, 3-methyl-cholanthrene, ethanol or pregnenolone 16α-carbonitrile had little or no effect on the liver microsomal metabolism of TMA to TMAO or DMA. Changing the atmosphere in the incubation vessel from 20% oxygen/80% nitrogen (air) to 100% oxygen had a selective stimulatory effect on the N-oxygenation of TMA but did not affect TMA N-demethylation. In addition, the K m for TMA N-demethylation was 5-fold higher than for the N-oxygenation reaction. The results of these studies suggest that the enzyme systems responsible for N-demethylation and N-oxygenation are different and that they are under different regulatory control. Carbon monoxide ( CO/O 2 = 80 20 ) had little or no inhibitory effect on either the N-demethylation or N-oxygenation of TMA by liver chromosomes from control or pregnenolone 16α-carbonitrile-treated rats. Additional studies indicated that methimazole, an inhibitor of FAD-containing monooxygenase (FMO), was a potent inhibitor of TMA oxidation. Preincubation of liver microsomes from control or pregnenolone 16α-carbonitrile-treated rats at 37° for 10 min without NADP(H) (a procedure that irreversibly inactivated FMO activity) resulted in > 95% inhibition of TMA N-demethylation and N-oxygenation, and this inhibition was prevented by including a NADPH-generating system in the preincubation medium (a procedure for preventing the thermal inactivation of FMO activity). The data suggest that FMOs are the major enzymes responsible for N-demethylation and N-oxygenation of TMA in rat liver microsomes.

Neurotoxins: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, 1,2,3,4-tetrahydroisoquinoline and 1-methyl-6,7-dihydroxy-tetrahydroisoquinoline as substrates for FAD-containing monooxygenase of porcine liver microsomes

The activity of FAD-containing monooxygenase (FMO) (EC 1.14.13.8) of porcine liver microsomes was examined with the neurotoxins, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 1,2,3,4-tetrahydroisoquinoline (TIQ) and 1-methyl-6,7-dihydroxy-tetrahydroisoquinoline (MDTIQ), as substrates. FMO catalyses these neurotoxins. The kinetic parameters of FMO for the neurotoxins and electron donors were determined. K m values for MPTP, TIQ and MDTIQ were determined to be 47 μM, 6.9 mM and 5.6 mM, respectively. The K m for the electron donor, NADPH, was variable from 31 to 200 μM depending on the substrate used. The activities of FMO for these neurotoxins were comparable with that for dimethylaniline.

Biosynthesis of methylguanidine in the hepatic microsomal fraction.

We have investigated various synthetic mechanisms for the production of methylguanidine, a potent uremic toxin, and reported a role for active oxygen in its biosynthesis from creatinine in studies using isolated hepatocytes. In this study, we turn our attention to the hepatic microsomes. Liver homogenates were made from rats, and various organelles were obtained by centrifugation and incubated with creatinine. The results show that methylguanidine synthesis occurs only in the microsomal fraction in the presence of nicotinamide adenine dinucleotide phosphate, reduced form. The microsomal activity is inhibited by the addition of methimazole, metyrapone, superoxide dismutase, catalase or dimethylsulfoxide. These results suggest that methylguanidine is synthesized from creatinine by microsomes, and at least 2 enzymes are involved, an FAD-containing monooxygenase and a P-450-dependent oxidase based on the inhibitory effect of methimazole and metyrapone, respectively. Moreover, the inhibition by various scavengers of active oxygen suggests that active oxygen plays a role in the intermediate steps of the enzymatic reaction.