Microbial metabolism of the pyridine ring. The metabolism of pyridine-3,4-diol (3,4-dihydroxypyridine) by Agrobacterium sp

1. The first metabolic step in the biodegradation of 4-hydroxypyridine by an Agrobacterium sp. was hydroxylation to form pyridine-3,4-diol. 2. Extracts required 1mol of O(2) and 1mol of NADH or NADPH for the conversion of 4-hydroxypyridine into pyridine-3,4-diol, suggesting that the enzyme responsible, 4-hydroxypyridine-3-hydroxylase, was a mixed function mono-oxygenase. 3. After treatment with acidic (NH(4))(2)SO(4) the enzyme required FAD for activity; FMN and riboflavin would not substitute for FAD. 4. The rate of anaerobic reduction of FAD by NAD(P)H was increased more than tenfold in the presence of 4-hydroxypyridine, suggesting that the mechanism of hydroxylation was similar to that of other aromatic hydroxylases which are of the mono-oxygenase type. 5. The partially purified enzyme was extremely specific for its heterocyclic substrate but would utilize either NADH or NADPH. 6. 4-Hydroxypyridine-3-hydroxylase was strongly inhibited by high substrate concentration (above 0.5mm) especially below pH7.5. 8. The inflexion at pH8.4 in a pK(m) versus pH plot, together with strong inhibition by p-chloromercuribenzoate, suggested a role for thiol groups in substrate binding.     

Ecdysone 20-mono-oxygenase in the desert locust, Schistocerca gregaria.

The enzyme catalysing the hydroxylation of ecdysone to 20-hydroxyecdysone, ecdysone 20-mono-oxygenase (EC 1.14.99.22), was investigated in the Malpighian tubules of fifth-instar locusts, Schistocerca gregaria. Enzyme activity was optimal at 35 degrees C and pH 6.8-8.0. Under these conditions the mono-oxygenase exhibited an apparent Km for ecdysone of 7.1 X 10(-7) M, a maximal specific activity of 1.1 nmol/h per mg of protein and was competitively inhibited by 20-hydroxyecdysone with an apparent Ki of 6.3 X 10(-7) M. Enzyme activity was decreased in the presence of Ca2+, Mg2+, EDTA and non-ionic detergents. The Malpighian tubule ecdysone 20-mono-oxygenase was localized primarily in the subcellular fraction sedimenting at 7500 g and, on the basis of marker enzyme profiles, was assigned mainly to the mitochondria. NADPH was required for activity, although addition of NADH together with NADPH had a synergistic effect. NADP+-dependent isocitrate dehydrogenase (EC 1.1.1.42) and an energy-dependent NAD(P) transhydrogenase (EC 1.6.1.1.) appeared to be the major sources of reducing equivalents, with the contribution from the 'malic enzyme' (EC 1.1.1.40) being less important. The monooxygenase was characterized as a cytochrome P-450-containing mixed-function oxidase from the inhibition patterns with metyrapone, CO and cyanide; CO inhibition was reversible with monochromatic light at 450 nm. However, the ecdysone 20-mono-oxygenase shows much lower sensitivity to CO inhibition and to photodissociation of the CO-inhibited complex than do vertebrate cytochrome P-450-dependent hydroxylation systems. The concentration of cytochrome P-450 in the Malpighian tubule mitochondria was 30 pmol/mg of protein. The properties of the mono-oxygenase are discussed in relation to hydroxylation enzymes from other sources.     

Functional characterization and mechanism of action of recombinant human kynurenine 3-hydroxylase.

The mitochondrial outer membrane enzyme kynurenine 3-hydroxylase (K3H) is an NADPH-dependent flavin mono-oxygenase involved in the tryptophan pathway, where it catalyzes the hydroxylation of kynurenine. K3H was transiently expressed in COS-1 cells as a glutathione S-transferase (GST) fusion protein, and the pure recombinant protein (rec-K3H) was obtained with a specific activity of about 2000 nmol.min-1.mg-1. Rec-K3H was shown to have an optimum pH at 7.5, to use NADPH more efficiently than NADH, and to contain one molecule of non-covalently bound FAD per molecule of enzyme. The mechanism of the rec-K3H-catalyzed reaction was investigated by overall initial-rate measurements, and a random mechanism in which combination of the enzyme with one substrate does not influence its affinity for the other is proposed. Further kinetic studies revealed that K3H activity was inhibited by both pyridoxal phosphate and Cl-, and that NADPH-catalyzed oxidation occurred even in the absence of kynurenine if 3-hydroxykynurenine was present, suggesting an uncoupling effect of 3-hydroxykynurenine with peroxide formation. This observation could be of clinical interest, as peroxide formation could explain the neurotoxicity of 3-hydroxykynurenine in vivo.     

Reconstitution of CMP-N-acetylneuraminic acid hydroxylation activity using a mouse liver cytosol fraction and soluble cytochrome b5 purified from horse erythrocytes.

The hydroxylation of CMP-N-acetylneuraminic acid (CMP-NeuAc) in the formation of CMP-N-glycolylneuraminic acid requires several components which comprise an electron transport system. A protein, which replaces one of the components, was purified to homogeneity from a horse erythrocyte lysate. Based on its partial amino acid sequence and immunological cross-reactivity, this protein was identified as soluble cytochrome b5 lacking the membrane domain of microsomal cytochrome b5. The electron transport system involved in CMP-NeuAc hydroxylation was reconstituted, and then characterized using the purified horse soluble cytochrome b5 and a fraction from mouse liver cytosol. The hydroxylation reaction requires a reducing reagent, DTT being the most effective. Either NADH or NADPH was used as an electron donor, but the activity with NADPH amounted to about 74% of that with NADH. The hydroxylation was inhibited by salts and azide due to interruption of the electron transport from NAD(P)H to cytochrome b5 and in the terminal enzyme reaction, respectively.     

Recombinant lysine:N(6)-hydroxylase: effect of cysteine-->alanine replacements on structural integrity and catalytic competence

Recombinant lysine:N(6)-hydroxylase, rIucD, catalyzes the hydroxylation of L-lysine to its N(6)-hydroxy derivative, with NADPH and FAD serving as cofactors in the reaction. The five cysteine residues present in rIucD can be replaced, individually or in combination, with alanine without effecting a major change in the thermal stability, the affinity for L-lysine and FAD, as well as the k(cat) for mono-oxygenase activity of the protein. However, when the susceptibility to modification by either 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) or 2,6-dichlorophenol indophenol (DPIP) serves as the criterion for monitoring conformational change(s) in rIucD and its muteins, Cys146-->Ala and Cys166-->Ala substitutions are found to induce an enhancement in the reactivity of one of the protein's remaining cysteine residues with concomitant diminution of mono-oxygenase function. In addition, the systematic study of cysteine-->alanine replacement has led to the identification of rIucD's Cys166 as the exposed residue which is detectable during the reaction of the protein with DTNB but not with iodoacetate. Substitution of Cys51 of rIucD with alanine results in an increase in mono-oxygenase activity (approx. 2-fold). Such replacement, unlike those of other cysteine residues, also enables the covalent DPIP conjugate of the protein to accommodate FAD in its catalytic function. A possible role of rIucD's Cys51 in the modulation of its mono-oxygenase function is discussed.     

A stopped-flow kinetic study of soluble methane mono-oxygenase from Methylococcus capsulatus (Bath).

1. The roles of the three protein components of soluble methane mono-oxygenase were investigated by the use of rapid-reaction techniques. The transfer of electrons through the enzyme complex from NADH to methane/O2 was also investigated. 2. Electron transfer from protein C, the reductase component, to protein A, the hydroxylase component, was demonstrated. Protein C was shown to undergo a three-electron--one-electron catalytic cycle. The interaction of protein C with NADH was investigated. Reduction of protein C was shown to be rapid, and a charge-transfer interaction between reduced FAD and NAD+ was observed; this intermediate was also found in static titration experiments. Thus the binding of NADH, the reduction of protein C and the intramolecular transfer of electrons through protein C were shown to be much more rapid than the turnover rate of methane mono-oxygenase. 3. The rate of transfer of electrons from protein C to protein A was shown to be lower than the reduction of protein C but higher than the turnover rate of methane mono-oxygenase. Association of the proteins was not rate-limiting. The amount of protein A present in the system had a small effect on the rate of reduction of protein C, indicating some co-operativity between the two proteins. 4. Protein B was shown to prevent electron transfer between protein C and protein A in the absence of methane. On addition of saturating concentrations of methane electron transfer was restored. With saturating concentrations of methane and O2 the observed rate constant for the conversion of methane into methanol was 0.26 s-1 at 18 degrees C. 5. By the use of [2H4]methane it was demonstrated that C-H-bond breakage is likely to be the rate-limiting step in the conversion of methane into methanol.     

Oxidation of carbon monoxide and methane by Pseudomonas methanica.

The oxidation of carbon monoxide and methane by suspensions and ultrasonic extracts of Pseudomonas methanica was studied. A continuous assay for the oxidation of CO to CO2 was devised, using O2 and CO2 electrodes in combination. Stoicheiometries of CO-dependent CO2 formation, O2 consumption and NADH oxidation, and the partial stoicheiometries of methane-dependent NADH oxidation, suggest the involvement of a mono-oxygenase in these oxidations. Evidence is presented suggesting methane and CO oxidation are catalysed by a single enzyme system, distinct, at least in part, from the NADH oxidase present in extracts. Ethanol was able to provide the reductant necessary for CO oxidation by cell suspensions, though the metabolism of ethanol by P. methanica was found unlikely to result in substrate-level formation of NADH; the means whereby alcohol oxidation could supply reductant for the mono-oxygenase are discussed.     

Hydroxylation of indole by laboratory-evolved 2-hydroxybiphenyl 3-monooxygenase

Directed enzyme evolution of 2-hydroxybiphenyl 3-monooxygenase (HbpA; EC ) from Pseudomonas azelaica HBP1 resulted in an enzyme variant (HbpA(ind)) that hydroxylates indole and indole derivatives such as hydroxyindoles and 5-bromoindole. The wild-type protein does not catalyze these reactions. HbpA(ind) contains amino acid substitutions D222V and V368A. The activity for indole hydroxylation was increased 18-fold in this variant. Concomitantly, the K(d) value for indole decreased from 1.5 mm to 78 microm. Investigation of the major reaction products of HbpA(ind) with indole revealed hydroxylation at the carbons of the pyrrole ring of the substrate. Subsequent enzyme-independent condensation and oxidation of the reaction products led to the formation of indigo and indirubin. The activity of the HbpA(ind) mutant monooxygenase for the natural substrate 2-hydroxybiphenyl was six times lower than that of the wild-type enzyme. In HbpA(ind), there was significantly increased uncoupling of NADH oxidation from 2-hydroxybiphenyl hydroxylation, which could be attributed to the substitution D222V. The position of Asp(222) in HbpA, the chemical properties of this residue, and the effects of its substitution indicate that Asp(222) is involved in substrate activation in HbpA.     

Mechanism and regulation of the Two-component FMN-dependent monooxygenase ActVA-ActVB from Streptomyces coelicolor

The ActVA-ActVB system from Streptomyces coelicolor is a two-component flavin-dependent monooxygenase involved in the antibiotic actinorhodin biosynthesis. ActVB is a NADH:flavin oxidoreductase that provides a reduced FMN to ActVA, the monooxygenase that catalyzes the hydroxylation of dihydrokalafungin, the precursor of actinorhodin. In this work, using stopped-flow spectrophotometry, we investigated the mechanism of hydroxylation of dihydrokalafungin catalyzed by ActVA and that of the reduced FMN transfer from ActVB to ActVA. Our results show that the hydroxylation mechanism proceeds with the participation of two different reaction intermediates in ActVA active site. First, a C(4a)-FMN-hydroperoxide species is formed after binding of reduced FMN to the monooxygenase and reaction with O(2). This intermediate hydroxylates the substrate and is transformed to a second reaction intermediate, a C(4a)-FMN-hydroxy species. In addition, we demonstrate that reduced FMN can be transferred efficiently from the reductase to the monooxygenase without involving any protein.protein complexes. The rate of transfer of reduced FMN from ActVB to ActVA was found to be controlled by the release of NAD(+) from ActVB and was strongly affected by NAD(+) concentration, with an IC(50) of 40 microm. This control of reduced FMN transfer by NAD(+) was associated with the formation of a strong charge.transfer complex between NAD(+) and reduced FMN in the active site of ActVB. These results suggest that, in Streptomyces coelicolor, the reductase component ActVB can act as a regulatory component of the monooxygenase activity by controlling the transfer of reduced FMN to the monooxygenase.     

Xanthine:NAD+ oxidoreductase from embryo liver of hen Gallus gallus

1. Xanthine:NAD+ oxidoreductase from chick embryo liver is unconvertible to the O2-dependent form, as is the enzyme from the adult hen. The Km for NAD+ (approximately 3 microM) of the embryonic enzyme is equal to, and the Km for xanthine (approximately 5 microM) is 2.5-fold lower, when compared with respective Km values of the "adult" hen enzyme. The inhibition of embryonic enzyme by NADH begins at 10 microM NADH and attains 13% at 35 microM NADH (respective data for the "adult" enzyme: 50 microM and 20% at 80 microM NADH). 2. The course of hypoxanthine----xanthine----uric acid hydroxylation catalyzed by the embryonic and "adult" enzymes is similar, however the rate of the first reaction is 2-fold lower for the embryonic enzyme. Under conditions of the limited nutritional system in the developing chick embryo, the low rate of hypoxanthine hydroxylation may promote reutilization of hypoxanthine for nucleotide synthesis.     

Hydroxylation of p-Coumaric acid by illuminated chloroplasts. The role of superoxide.

1. Chloroplasts isolated from leaves of spinach-beet (Beta vulgaris L. ssp. vulgaris) do not catalyse the hydroxylation of p-coumaric acid in the dark unless a reductant (such as ascorbate, NADH or NADPH) is added. Superoxide dismutase has no effect on this reaction. 2. Illuminated chloroplasts catalyse the hydroxylation in the absence of added reductant. This reaction is completely inhibited by superoxide dismutase, but catalase has little effect. 3. Both hydroxylation in the light and hydroxylation in the dark in the presence of reductants are inhibited by diethyldithiocarbamate, EDTA, cyanide and 2-mercaptoethanol. 4. It is proposed that O-2- generated by illuminated chloroplasts is involved in the provision of a reductant to the enzyme phenolase.     

2-Aminobenzoyl-CoA monooxygenase/reductase, a novel type of flavoenzyme. Purification and some properties of the enzyme

A novel aerobic mechanism of 2-aminobenzoate metabolism was proposed in a denitrifying Pseudomonas species. 2-Aminobenzoic acid is activated in a coenzyme-A-ligase reaction to 2-aminobenzoyl-CoA and this intermediate is dearomatized by a unique enzyme, tentatively named 2-aminobenzoyl-CoA monooxygenase/reductase. This paper describes the purification and some molecular, kinetic and spectral properties of this flavoenzyme which catalyzes the hydroxylation and reduction of 2-aminobenzoyl-CoA to an unknown non-aromatic compound. 2-Aminobenzoyl-CoA monooxygenase/reductase was purified 25-fold to a specific activity of 25 mumol.min-1.mg-1 protein using ammonium sulfate precipitation, DEAE-cellulose anion-exchange, hydroxylapatite and Mono Q FPLC anion-exchange chromatography. Superose 6 gel filtration for estimation of molecular mass resulted in one symmetrical protein peak corresponding to a molecular mass of 170 kDa. Several experimental data suggest that the protein is probably an alpha 2 dimer; however, it may exist in three dimeric forms, alpha alpha, alpha alpha' and alpha' alpha', where alpha' may be a subunit with a different conformation. Approximately 2 mol noncovalently bound FAD/mol enzyme was found, which in the absence of O2 was reduced by NADH. The enzyme was specific for the substrates 2-aminobenzoyl-CoA (Km less than or equal to 25 microM) and O2 (Km less than or equal to 5 microM), but less specific for the reduced pyridine nucleotides NADH (Km = 42 microM) or NADPH [Km = 500 microM; Vmax (NADH)/Vmax (NADPH) = 1.7:1]. The turnover number was 4250 min-1. The enzyme also reduced N-ethylmaleimide and maleimide with NAD(P)H. The substrate, the products and the reaction stoichiometry are described in two following papers.     

Combined participation of hydroxylase active site residues and effector protein binding in a para to ortho modulation of toluene 4-monooxygenase regiospecificity

Toluene 4-monooxygenase (T4MO) is a diiron hydroxylase that exhibits high regiospecificity for para hydroxylation. This fidelity provides the basis for an assessment of the interplay between active site residues and protein complex formation in producing an essential biological outcome. The function of the T4MO catalytic complex (hydroxylase, T4moH, and effector protein T4moD) is evaluated with respect to effector protein concentration, the presence of T4MO electron-transfer components (Rieske ferredoxin, T4moC, and NADH oxidoreductase), and use of mutated T4moH isoforms with different hydroxylation regiospecificities. Steady-state kinetic analyses indicate that T4moC and T4moD form complexes of similar affinity with T4moH. At low T4moD concentrations, the steady-state hydroxylation rate is linearly dependent on T4moD-T4moH complex formation, whereas regiospecificity and the coupling efficiency between NADH consumption and hydroxylation are associated with intrinsic properties of the T4moD-T4moH complex. The optimized complex gives both efficient coupling and high regiospecificity with p-cresol representing >96% of total products from toluene. Similar coupling and regiospecificity for para hydroxylation are obtained with T3buV (an effector protein from a toluene 3-monooxygenase), demonstrating that effector protein binding does not uniquely determine or alter the regiospecificity of toluene hydroxylation. The omission of T4moD causes an approximately 20-fold decrease in hydroxylation rate, nearly complete uncoupling, and a decrease in regiospecificity so that p-cresol represents approximately 60% of total products. Similar shifts in regiospecificity are observed in oxidations of alternative substrates in the absence or upon the partial removal of either T4moD or T3buV from toluene oxidations. The mutated T4moH isoforms studied have apparent V(max)/K(M) specificities differing by approximately 2-4-fold and coupling efficiencies ranging from 88% to 95%, indicating comparable catalytic function, but also exhibit unique regiospecificity patterns for all substrates tested, suggesting unique substrate binding preferences within the active site. The G103L isoform has enhanced selectivity for ortho hydroxylation with all substrates tested except nitrobenzene, which gives only m-nitrophenol. The regiospecificity of the G103L isoform is comparable to that observed from naturally occurring variants of the toluene/benzene/o-xylene monooxygenase subfamily. Evolutionary and mechanistic implications of these findings are considered.     

A new UV method for serum gamma-glutamyltransferase assay using recombinant 4-aminobenzoate hydroxylase as a coupling enzyme.

4-aminobenzoate hydroxylase (4ABH) is a flavin-dependent monooxygenase that catalyzes the decarboxylative hydroxylation of 4-aminobenzoate to 4-hydroxyaniline. For use as a clinical reagent, the gene encoding 4ABH from Agaricus bisporus was cloned by the RACE method. Also, the cDNA encoding 4ABH was expressed in Escherichia coli cells as a fusion protein with glutathione S-transferase (GST). The expressed GST-4ABH fusion protein (recombinant 4ABH) in the soluble fraction exhibits decarboxylative hydroxylation and additional NADH oxidation activities.We investigated a new ultraviolet spectrometric method for determining serum gamma-glutamyltransferase (gamma-GT) using recombinant 4ABH as a coupling enzyme. The principle of the method is as follows. Using gamma-glutamyl-3-choloro-4-aminobenzoate (L-gamma-glu-PAClBA) and glycylglycine as the donor and acceptor substrates, 3-choloro-4-aminobenzoate (PAClBA) is formed by the catalysis of serum gamma-GT. PAClBA is stoichiometrically converted to 3-choloro-4-hydroxyaniline (PHClA) and NAD(+) by 4ABH and NADH. However, NADH oxidation results in a high reagent blank, which is considered as a drawback for use as a clinical reagent.Using recombinant 4ABH, we examined the effects of pH and detergents on these two activities, and found that several detergents suppress the additional NADH oxidation activity with little or no effect on hydroxylation activity. The results indicate a promising approach to establishing an ultraviolet spectrophotometric method for determining serum gamma-GT activity using L-gamma-glu-PAClBA as the donor substrate and recombinant 4ABH as a coupling enzyme.     

Elicitor induction of a microsomal 5-O-(4-coumaroyl)shikimate 3'-hydroxylase in parsley cell suspension cultures

Microsomal preparations from parsley cell suspension cultures challenged with an elicitor from Phytophthora megasperma f.sp. glycinea (Pmg) catalyze the formation of trans-5-O-caffeoylshikimate from trans-5-O-(4-coumaroyl)shikimate. Neither the cis isomer nor free 4-coumarate, 4-coumaroyl-CoA, or 5-O-(4-coumaroyl)quinate are substrates for this enzyme. The reaction is strictly dependent on NADPH as a reducing cofactor and on molecular oxygen. NADH, ascorbic acid, and 6,7-dimethyl-5,6,7,8-tetrahydropterine cannot substitute for NADPH. However, NADH enhances enzyme activity observed in the presence of NADPH. Cytochrome c and carbon monoxide inhibit the hydroxylation reaction, suggesting a cytochrome P-450-dependent mixed-function monooxygenase.     

Electron transfer reactions in the soluble methane monooxygenase of Methylococcus capsulatus (Bath)

Aerobic stopped-flow experiments have confirmed that component C is the methane monooxygenase component responsible for interaction with NADH. Reduction of component C by NADH is not the rate-limiting step for component C in the methane monooxygenase reaction. Removal and reconstitution of the redox centres of component C suggest a correlation between the presence of the FAD and Fe2S2 redox centres and NADH: acceptor reductase activity and methane monooxygenase activity respectively, consistent with the order of electron flow: NADH----FAD----Fe2S2----component A. This order suggests that component C functions as a 2e-1/1e-1 transformase, splitting electron pairs from NADH for transfer to component A via the one-electron-carrying Fe2S2 centre. Electron transfer has been demonstrated between the reductase component, component C and the oxygenase component, component A, of the methane monooxygenase complex from Methylococcus capsulatus (Bath) by three separate methods. This intermolecular electron transfer step is not rate-determining for the methane monooxygenase reaction. Intermolecular electron transfer was independent of component B, the third component of the methane monooxygenase. Component B is required to switch the oxidase activity of component A to methane mono-oxygenase activity, suggesting that the role of component B is to couple substrate oxidation to electron transfer, via the methane monooxygenase components.     

A steady-state kinetic study of the reaction catalysed by the secondary-amine mono-oxygenase of Pseudomonas aminovorans.

1. Secondary-amine mono-oxygenase (proposed EC group 1.14.99.-) was partially purified from trimethylamine-grown Pseudomonas aminovorans by (NH4)2SO4 fractionation, gel filtration, hydrophobic chromatography on 5-aminopentylamino-Sepharose, and affinity chromatography on Sepharose-bound NADH. 2. Some problems in the affinity-chromatography step are discussed. 3. A steady-state kinetic analysis varying substrate, oxygen and electron-donor concentrations was performed, which, over the concentration range studied, gave a series of families of approximately parallel double-reciprocal plots. From secondary and tertiary plots, Michaelis constants of 0.160 mM, 0.086 mM and 0.121 mM were obtained for dimethylamine, NADPH and oxygen respectively. 4. Product-inhibition studies supported the postulated Hexa Uni Ping Pong (triple-transfer) reaction mechanism.     

Regioselective synthesis of 1,3,5-adamantanetriol from 1,3-adamantanediol using Kitasatospora cells

Washed cells (62?mg) of Kitasatospora sp. GF12 in 4?ml buffer (pH 7) catalyzed the regioselective hydroxylation of 60?mM 1,3-adamantanediol [1,3-ad(OH)(2)] to 30.9?mM 1,3,5-adamantanetriol [1,3,5-ad(OH)(3)] over 120?h at 24?°C. Glycerol at 400?mM was added to the reaction mixture to recycle the intracellular NADH/NADPH. Whole cells of GF12, also catalyzed the hydroxylation of 10?mM 1-adamantanol (1-adOH), to 3.6?mM 1,3,5-ad(OH)(3).     

Kinetics of benzo(a)pyrene hydroxylation at varying concentrations of NADPH and NADH in liver microsomes of nontreated and beta-naphthoflavone-treated C57BL/2 and DBA/2 mice

The biphasic kinetics of hydroxylation of benzo(a)pyrene (B(a)P) dependent on the concentration of either NADPH or NADH has been observed in DBA/2 (AhdAhd) but not in C57BL/6 (AhbAhb) beta-naphthoflavone-treated mice. On the other hand, in nontreated mice, this biphasic kinetics has been observed in both strains of mice. This shows that characteristics of biphasic kinetics differentiate Ahb from Ahd mice only after treatment with methylcholanthrene-type of inducers. The discussed biphasic kinetics was more regular and distinct in the NADH-dependent reaction as compared with NADPH-dependent hydroxylation of B(a)P. It is suggested that the NADH-specific pathway of electron transport to cytochrome P-450 is necessary for the occurrence of this effect both in NADH- and NADPH supported reaction.     

Chlorzoxazone hydroxylation in microsomes and hepatocytes from cytochrome P450 oxidoreductase‐null mice

Previous studies have demonstrated that the NADH‐dependent cytochrome b₅ electron transfer pathway can support some cytochrome P450 monooxygenases in vitro in the absence of their normal redox partner, NADPH‐cytochrome P450 oxidoreductase. However, the ability of this pathway to support P450 activity in whole cells and in vivo remains unresolved. To address this question, liver microsomes and hepatocytes were prepared from hepatic cytochrome P450 oxidoreductase‐null mice and chlorzoxazone hydroxylation, a reaction catalyzed primarily by cytochrome P450 2E1, was evaluated. As expected, NADPH‐supported chlorzoxazone hydroxylation was absent in liver microsomes from oxidoreductase‐null mice, whereas NADH‐supported activity was about twofold higher than that found in normal (wild‐type) liver microsomes. This greater activity in oxidoreductase‐null microsomes could be attributed to the fourfold higher level of CYP2E1 and 1.4‐fold higher level of cytochrome b₅. Chlorzoxazone hydroxylation in hepatocytes from oxidoreductase‐null mice was about 5% of that in hepatocytes from wild‐type mice and matched the results obtained with wild‐type microsomes, where activity obtained with NADH was about 5% of that obtained when both NADH and NADPH were included in the reaction mixture. These results argue that the cytochrome b₅ electron transfer pathway can support a low but measurable level of CYP2E1 activity under physiological conditions. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:357–363, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20299