Remarkable aliphatic hydroxylation by the diiron enzyme toluene 4-monooxygenase in reactions with radical or cation diagnostic probes norcarane, 1,1-dimethylcyclopropane, and 1,1-diethylcyclopropane

Toluene 4-monooxygenase (T4MO) catalyzes the hydroxylation of toluene to yield 96% p-cresol. This diiron enzyme complex was used to oxidize norcarane (bicyclo[4.1.0]heptane), 1,1-dimethylcyclopropane, and 1,1-diethylcyclopropane, substrate analogues that can undergo diagnostic reactions upon the production of transient radical or cationic intermediates. Norcarane closely matches the shape and volume of the natural substrate toluene. Reaction of isoforms of the hydroxylase component of T4MO (T4moH) with different regiospecificities for toluene hydroxylation (k(cat) approximately 1.9-2.3 s(-)(1) and coupling efficiency approximately 81-96%) revealed similar catalytic parameters for norcarane oxidation (k(cat) approximately 0.3-0.5 s(-)(1) and coupling efficiency approximately 72%). The products included variable amounts of the un-rearranged isomeric norcaranols and cyclohex-2-enyl methanol, a product attributed to rearrangement of a radical oxidation intermediate. A ring-expansion product derived from the norcaranyl C-2 cation, cyclohept-3-enol, was not produced by either the natural enzyme or any of the T4moH isoforms tested. Comparative studies of 1,1-dimethylcyclopropane and 1,1-diethylcyclopropane, diagnostic substrates with differences in size and with approximately 50-fold slower k(cat) values, gave products consistent with both radical rearrangement and cation ring expansion. Examination of the isotopic enrichment of the incorporated O-atoms for all products revealed high-fidelity incorporation of an O-atom from O(2) in the un-rearranged and radical-rearranged products, while the O-atom found in the cation ring-expansion products was predominantly obtained by reaction with H(2)O. The results show a divergence of radical and cation pathways for T4moH-mediated hydroxylation that can be dissected by diagnostic substrate probe rearrangements and by changes in the source of oxygen used for substrate oxygenation.     

Reaction mechanisms of non-heme diiron hydroxylases characterized in whole cells

Whole cells expressing the non-heme diiron hydroxylases AlkB and toluene 4-monooxygenase (T4MO) were used to probe enzyme reaction mechanisms. AlkB catalyzes the hydroxylation of the radical clock substrates bicyclo[4.1.0]heptane (norcarane), spirooctane and 1,1-diethylcyclopropane, and does not catalyze the hydroxylation of the radical clocks 1,1-dimethylcyclopropane or 1,1,2,2-tetramethylcyclopropane. The hydroxylation of norcarane yields a distribution of products consistent with an "oxygen-rebound" mechanism for the enzyme in both the wild type Pseudomonas putida GPo1 and AlkB from P. putida GPo1 expressed in Escherichia coli. Evidence for the presence of a substrate-based radical during the reaction mechanism is clear. With norcarane, the lifetime of that radical varies with experimental conditions. Experiments with higher substrate concentrations yield a shorter radical lifetime (approximately 1 ns), while experiments with lower substrate concentrations yield a longer radical lifetime (approximately 19 ns). Consistent results were obtained using either wild type or AlkB-equipped host organisms using either "resting cell" or "growing cell" approaches. T4MO expressed in E. coli also catalyzes the hydroxylation of norcarane with a radical lifetime of approximately 0.07 ns. No radical lifetime dependence on substrate concentration was seen. Results from experiments with diethylcyclopropane, spirooctane, dimethylcyclopropane, and diethylcyclopropane are consistent with a restricted active site for AlkB.     

Resonance Raman studies of the stoichiometric catalytic turnover of a substrate-stearoyl-acyl carrier protein delta(9) desaturase complex

Resonance Raman spectroscopy has been used to study the effects of substrate binding (stearoyl-acyl carrier protein, 18:0-ACP) on the diferric centers of Ricinus communis 18:0-ACP Delta(9) desaturase. These studies show that complex formation produces changes in the frequencies of nu(s)(Fe-O-Fe) and nu(as)(Fe-O-Fe) consistent with a decrease in the Fe-O-Fe angle from approximately 123 degrees in the oxo-bridged diferric centers of the as-isolated enzyme to approximately 120 degrees in oxo-bridged diferric centers of the complex. Analysis of the shifts in nu(s)(Fe-O-Fe) and nu(as)(Fe-O-Fe) as a function of 18:0-ACP concentration also suggests that 4e(-)-reduced Delta9D containing two diferrous centers has a higher affinity for 18:0-ACP than resting Delta9D containing two diferric centers. Catalytic turnover of a stoichiometric complex of 18:0-ACP and Delta9D was used to investigate whether an O-atom from O(2) would be incorporated into a bridging position of the resultant mu-oxo-bridged diferric centers during the desaturation reaction. Upon formation of approximately 70% yield of 18:1-ACP product in the presence of (18)O(2), no incorporation of an (18)O atom into the mu-oxo bridge position was detected. The result with 18:0-ACP Delta(9) desaturase differs from that obtained during the tyrosyl radical formation reaction of the diiron enzyme ribonucleotide reductase R2 component, which proceeds with incorporation of an O-atom from O(2) into the mu-oxo bridge of the resting diferric site. The possible implications of these results for the O-O bond cleavage reaction and the nature of intermediates formed during Delta9D catalysis are discussed.     

Nucleotide sequence analysis of genes encoding a toluene/benzene-2-monooxygenase from Pseudomonas sp. strain JS150.

It was previously shown by others that Pseudomonas sp. strain JS150 metabolizes benzene and alkyl- and chloro-substituted benzenes by using dioxygenase-initiated pathways coupled with multiple downstream metabolic pathways to accommodate catechol metabolism. By cloning genes encoding benzene-degradative enzymes, we found that strain JS150 also carries genes for a toluene/benzene-2-monooxygenase. The gene cluster encoding a 2-monooxygenase and its cognate regulator was cloned from a plasmid carried by strain JS150. Oxygen (18O2) incorporation experiments using Pseudomonas aeruginosa strains that carried the cloned genes confirmed that toluene hydroxylation was catalyzed through an authentic monooxygenase reaction to yield ortho-cresol. Regions encoding the toluene-2-monooxygenase and regulatory gene product were localized in two regions of the cloned fragment. The nucleotide sequence of the toluene/benzene-2-monooxygenase locus was determined. Analysis of this sequence revealed six open reading frames that were then designated tbmA, tbmB, tbmC, tbmD, tbmE, and tbmF. The deduced amino acid sequences for these genes showed the presence of motifs similar to well-conserved functional domains of multicomponent oxygenases. This analysis allowed the tentative identification of two terminal oxygenase subunits (TbmB and TbmD) and an electron transport protein (TbmF) for the monooxygenase enzyme. In addition to these gene products, all the tbm polypeptides shared significant homology with protein components from other bacterial multicomponent monooxygenases. Overall, the tbm gene products shared greater similarity with polypeptides from the phenol hydroxylases of Pseudomonas putida CF600, P35X, and BH than with those from the toluene monooxygenases of Pseudomonas mendocina KR1 and Burkholderia (Pseudomonas) pickettii PKO1. The relationship found between the phenol hydroxylases and a toluene-2-monooxygenase, characterized in this study for the first time at the nucleotide sequence level, suggested that DNA probes used for surveys of environmental populations should be carefully selected to reflect DNA sequences corresponding to the metabolic pathway of interest.     

Crystallographic analysis of active site contributions to regiospecificity in the diiron enzyme toluene 4-monooxygenase

Crystal structures of toluene 4-monooxygenase hydroxylase in complex with reaction products and effector protein reveal active site interactions leading to regiospecificity. Complexes with phenolic products yield an asymmetric μ-phenoxo-bridged diiron center and a shift of diiron ligand E231 into a hydrogen bonding position with conserved T201. In contrast, complexes with inhibitors p-NH(2)-benzoate and p-Br-benzoate showed a μ-1,1 coordination of carboxylate oxygen between the iron atoms and only a partial shift in the position of E231. Among active site residues, F176 trapped the aromatic ring of products against a surface of the active site cavity formed by G103, E104 and A107, while F196 positioned the aromatic ring against this surface via a π-stacking interaction. The proximity of G103 and F176 to the para substituent of the substrate aromatic ring and the structure of G103L T4moHD suggest how changes in regiospecificity arise from mutations at G103. Although effector protein binding produced significant shifts in the positions of residues along the outer portion of the active site (T201, N202, and Q228) and in some iron ligands (E231 and E197), surprisingly minor shifts (<1 ?) were produced in F176, F196, and other interior residues of the active site. Likewise, products bound to the diiron center in either the presence or absence of effector protein did not significantly shift the position of the interior residues, suggesting that positioning of the cognate substrates will not be strongly influenced by effector protein binding. Thus, changes in product distributions in the absence of the effector protein are proposed to arise from differences in rates of chemical steps of the reaction relative to motion of substrates within the active site channel of the uncomplexed, less efficient enzyme, while structural changes in diiron ligand geometry associated with cycling between diferrous and diferric states are discussed for their potential contribution to product release.     

Protein Engineering of Toluene Monooxygenases for Synthesis of Chiral Sulfoxides

Enantiopure sulfoxides are valuable asymmetric starting materials and are important chiral auxiliaries in organic synthesis. Toluene monooxygenases (TMOs) have been shown previously to catalyze regioselective hydroxylation of substituted benzenes and phenols. Here we show that TMOs are also capable of performing enantioselective oxidation reactions of aromatic sulfides. Mutagenesis of position V106 in the α-hydroxylase subunit of toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 and the analogous position I100 in toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1 improved both rate and enantioselectivity. Variant TomA3 V106M of TOM oxidized methyl phenyl sulfide to the corresponding sulfoxide at a rate of 3.0 nmol/min/mg protein compared with 1.6 for the wild-type enzyme, and the enantiomeric excess (pro-S) increased from 51% for the wild type to 88% for this mutant. Similarly, T4MO variant TmoA I100G increased the wild-type oxidation rate by 1.7-fold, and the enantiomeric excess rose from 86% to 98% (pro-S). Both wild-type enzymes showed lower activity with methyl para-tolyl sulfide as a substrate, but the improvement in the activity and enantioselectivity of the mutants was more dramatic. For example, T4MO variant TmoA I100G oxidized methyl para-tolyl sulfide 11 times faster than the wild type did and changed the selectivity from 41% pro-R to 77% pro-S. A correlation between regioselectivity and enantioselectivity was shown for TMOs studied in this work. Using in silico homology modeling, it is shown that residue I100 in T4MO aids in steering the substrate into the active site at the end of the long entrance channel. It is further hypothesized that the main function of V106 in TOM is the proper positioning or docking of the substrate with respect to the diiron atoms. The results from this work suggest that when the substrate is not aligned correctly in the active site, the oxidation rate is decreased and enantioselectivity is impaired, resulting in products with both chiral configurations.     

Redox and functional analysis of the Rieske ferredoxin component of the toluene 4-monooxygenase

Toluene 4-monooxygenase catalyzes the NADH- and O2-dependent hydroxylation of toluene to form p-cresol. The four-protein complex consists of a diiron hydroxylase, an oxidoreductase, a catalytic effector protein, and a Rieske-type ferredoxin (T4moC). Phylogenetic analysis suggests that T4moC is part of a clade specialized for reaction with diiron hydroxylases, possibly reflected in the conservation of W69, whose indole side chain makes close contacts with a bridging sulfide. In order to further investigate the possible origins of this specialization, T4moC, mutated variants of T4moC, and three other purified ferredoxins (the Thermus Rieske protein, the Burkholderia cepacia Rieske-type biphenyl dioxygenase ferredoxin BphF, and the Ralstonia pickettii PK01 toluene monooxygenase TbuB, the Rieske-type ferredoxin from another diiron monooxygenase complex) were studied by redox potential measurements and their ability to complement the catalytic function of the reconstituted toluene 4-monooxygenase complex. A saturation mutagenesis of T4moC W69 indicates that an aromatic residue may modulate the redox potential and is also necessary for activity and/or stability. The redox potential of T4moC was determined to be -173 mV, W69F T4moC was -139 mV, and TbuB was -150 mV. For comparison, BphF had a redox potential of -157 mV [Couture et al. (2001) Biochemistry 40, 84-92]. Of these ferredoxins, all except BphF were able to provide catalytic activity. Given the range in redox potentials observed in the active ferredoxins, shape and electrostatics are strongly implicated in the catalytic specialization. Mutagenesis of other T4moC surface residues gave further insight into possible origins of catalytic specialization. Thus R65A T4moC gave an alteration in apparent KM only, while D82A/D83A T4moC gave alterations in both apparent kcat and KM. Since the different catalytic results were obtained by mutagenesis of residues lying on different sides of the protein adjacent to the [2Fe-2S] cluster, the results suggest that two different faces of T4moC may be involved in protein-protein interactions during catalysis.     

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.     

Threonine 201 in the diiron enzyme toluene 4-monooxygenase is not required for catalysis

The diiron enzyme toluene 4-monooxygenase from Pseudomonas mendocina KR1 catalyzes the NADH- and O(2)-dependent hydroxylation of toluene. A combination of sequence alignments and spectroscopic studies indicate that T4MO has an active site structure closely related to the crystallographically characterized methane monooxygenase hydroxylase. In the methane monooxygenase hydroxylase, active site residue T213 has been proposed to participate in O(2) activation by analogy to certain proposals made for cytochrome P450. In this work, mutagenesis of the comparable residue in the toluene 4-monooxygenase hydroxylase, T201, has been used to investigate the role of an active site hydroxyl group in catalysis. Five isoforms (T201S, T201A, T201G, T201F, and T201K) that retain catalytic activity based on an in vivo indigo formation assay were identified, and detailed characterizations of the purified T201S, T201A, and T201G variants are reported. These isoforms have k(cat) values of 1.2, 1.0, and 0.6 s(-)(1), respectively, and k(cat)/K(M) values that vary by only approximately 4-fold relative to that of the native isoform. Moreover, these isoforms exhibit 80-90% coupling efficiency, which also compares favorably to the >94% coupling efficiency determined for the native isoform. For the T201S, T201A, and T201G isoforms, the regiospecificity of toluene hydroxylation was nearly identical to that of the natural isoform, with p-cresol representing 90-95% of the total product distribution. In contrast, the T201F isoform caused a substantial shift in the product distribution, and gave o- and p-cresol in a 1:1 ratio. In addition, the amount of benzyl alcohol was increased approximately 10-fold with the T201F isoform. For reaction with p-xylene, previous studies have shown that the native isoform reacted to give 4-methybenzyl alcohol and 2, 5-dimethylphenol in a 4:1 ratio [Pikus, J. D., Studts, J. M., McClay, K., Steffan, R. J., and Fox, B. G. (1997) Biochemistry 36, 9283-9289]. For comparison, the T201S, T201A, and T201F isoforms gave a slightly relaxed 3:1 ratio of these products, while the T201G isoform gave a dramatically relaxed 1:1 ratio. On the basis of these studies, we conclude that the hydroxyl group of T201 is not essential to maintaining the turnover rate or the coupling of the toluene 4-monooxygenase complex. However, changing the volume occupied by the side chain at the position of T201 can lead to alterations in the regiospecificity of the hydroxylation, presumably by producing different orientations for substrate binding during catalysis.     

Multiple roles of component proteins in bacterial multicomponent monooxygenases: phenol hydroxylase and toluene/o-xylene monooxygenase from Pseudomonas sp. OX1

Phenol hydroxylase (PH) and toluene/o-xylene monooxygenase (ToMO) from Pseudomonas sp. OX1 require three or four protein components to activate dioxygen for the oxidation of aromatic substrates at a carboxylate-bridged diiron center. In this study, we investigated the influence of the hydroxylases, regulatory proteins, and electron-transfer components of these systems on substrate (phenol; NADH) consumption and product (catechol; H(2)O(2)) generation. Single-turnover experiments revealed that only complete systems containing all three or four protein components are capable of oxidizing phenol, a major substrate for both enzymes. Under ideal conditions, the hydroxylated product yield was ～50% of the diiron centers for both systems, suggesting that these enzymes operate by half-sites reactivity mechanisms. Single-turnover studies indicated that the PH and ToMO electron-transfer components exert regulatory effects on substrate oxidation processes taking place at the hydroxylase actives sites, most likely through allostery. Steady state NADH consumption assays showed that the regulatory proteins facilitate the electron-transfer step in the hydrocarbon oxidation cycle in the absence of phenol. Under these conditions, electron consumption is coupled to H(2)O(2) formation in a hydroxylase-dependent manner. Mechanistic implications of these results are discussed.     

Solution structure of the toluene 4-monooxygenase effector protein (T4moD)

Toluene 4-monooxygenase (T4MO) from Pseudomonas mendocina catalyzes the NADH- and O(2)-dependent hydroxylation of toluene to form p-cresol. The complex consists of an NADH oxidoreductase (T4moF), a Rieske ferredoxin (T4moC), a diiron hydroxylase [T4moH, with (alphabetagamma)(2) quaternary structure], and a catalytic effector protein (T4moD). The solution structure of the 102-amino acid T4moD effector protein has been determined from 2D and 3D (1)H, (13)C, and (15)N NMR spectroscopic data. The structural model was refined through simulated annealing by molecular dynamics in torsion angle space (DYANA software) with input from 1467 experimental constraints, comprising 1259 distance constraints obtained from NOEs, 128 dihedral angle constraints from J-couplings, and 80 hydrogen bond constraints. Of 60 conformers that met the acceptance criteria, the 20 that best satisfied the input constraints were selected to represent the solution structure. With exclusion of the ill-defined N- and C-terminal segments (Ser1-Asn11 and Asp99-Met102), the atomic root-mean-square deviation for the 20 conformers with respect to the mean coordinates was 0.71 A for the backbone and 1.24 A for all non-hydrogen atoms. The secondary structure of T4moD consists of three alpha-helices and seven beta-strands arranged in an N-terminal betaalphabetabeta and a C-terminal betaalphaalphabetabetabeta domain topology. Although the published NMR structures of the methane monooxygenase effector proteins from Methylosinus trichosporium OB3b and Methylococcus capsulatus (Bath) have a similar secondary structure topology, their three-dimensional structures differ from that of T4moD. The major differences in the structures of the three effector proteins are in the relative orientations of the two beta-sheets and the interactions between the alpha-helices in the two domains. The structure of T4moD is closer to that of the methane monooxygenase effector protein from M. capsulatus (Bath) than that from M. trichosporium OB3b. The specificity of T4moD as an effector protein was investigated by replacing it in reconstituted T4MO complexes with effector proteins from monooxygenases from other bacterial species: Pseudomonas pickettii PKO1 (TbuV, toluene 3-monooxygenase); Pseudomonas species JS150 (TbmC, toluene 2-monooxygenase); and Burkeholderia cepacia G4 (S1, toluene 2-monooxygenase). The results showed that the closely related TbuV effector protein (55% sequence identity) provided partial activation of the complex, whereas the more distantly related TbmC (34% sequence identity) and S1 (29% sequence identity) did not. The (1)H NMR chemical shifts of the side-chain amide protons of Asn34, a conserved, structurally relevant amino acid, were found to be similar in spectra of effector proteins T4moD and TbuV but not in the spectrum of TbmC. This suggests that the region around Asn34 may be involved in structural aspects contributing to functional specificity.     

Purification and characterization of 2,4,6-trichlorophenol-4-monooxygenase, a dehalogenating enzyme from Azotobacter sp. strain GP1.

The enzyme which catalyzes the dehalogenation of 2,4,6-trichlorophenol (TCP) was purified to apparent homogeneity from an extract of TCP-induced cells of Azotobacter sp. strain GP1. The initial step of TCP degradation in this bacterium is inducible by TCP; no activity was found in succinate-grown cells or in phenol-induced cells. NADH, flavin adenine dinucleotide, and O2 are required as cofactors. As reaction products, 2,6-dichlorohydroquinone and Cl- ions were identified. Studies of the stoichiometry revealed the consumption of 2 mol of NADH plus 1 mol of O2 per mol of TCP and the formation of 1 mol of Cl- ions. No evidence for membrane association or for a multicomponent system was obtained. Molecular masses of 240 kDa for the native enzyme and 60 kDa for the subunit were determined, indicating a homotetrameric structure. Cross-linking studies with dimethylsuberimidate were consistent with this finding. TCP was the best substrate for 2,4,6-trichlorophenol-4-monooxygenase (TCP-4-monooxygenase). The majority of other chlorophenols converted by the enzyme bear a chloro substituent in the 4-position. 2,6-Dichlorophenol, also accepted as a substrate, was hydroxylated in the 4-position to 2,6-dichlorohydroquinone in a nondehalogenating reaction. NADH and O2 were consumed by the pure enzyme also in the absence of TCP with simultaneous production of H2O2. The NH2-terminal amino acid sequence of TCP-4-monooxygenase from Azotobacter sp. strain GP1 revealed complete identity with the nucleotide-derived sequence from the analogous enzyme from Pseudomonas pickettii and a high degree of homology with two nondehalogenating monooxygenases. The similarity in enzyme properties and the possible evolutionary relatedness of dehalogenating and nondehalogenating monooxygenases are discussed.     

Trichloroethylene oxidation by purified toluene 2-monooxygenase: products, kinetics, and turnover-dependent inactivation.

Trichloroethylene is oxidized by several types of nonspecific bacterial oxygenases. Toluene 2-monooxygenase from Burkholderia cepacia G4 is implicated in trichloroethylene oxidation and is uniquely suggested to be resistant to turnover-dependent inactivation in vivo. In this work, the oxidation of trichloroethylene was studied with purified toluene 2-monooxygenase. All three purified toluene 2-monooxygenase protein components and NADH were required to reconstitute full trichloroethylene oxidation activity in vitro. The apparent Km and Vmax were 12 microM and 37 nmol per min per mg of hydroxylase component, respectively. Ten percent of the full activity was obtained when the small-molecular-weight enzyme component was omitted. The stable oxidation products, accounting for 84% of the trichloroethylene oxidized, were carbon monoxide, formic acid, glyoxylic acid, and covalently modified oxygenase proteins that constituted 12% of the reacted [14C]trichloroethylene. The stable oxidation products may all derive from the unstable intermediate trichloroethylene epoxide that was trapped by reaction with 4-(p-nitrobenzyl)pyridine. Chloral hydrate and dichloroacetic acid were not detected. This finding differs from that with soluble methane monooxygenase and cytochrome P-450 monooxygenase, which produce chloral hydrate. Trichloroethylene-dependent inactivation of toluene 2-monooxygenase activity was observed. All of the protein components were covalently modified during the oxidation of trichloroethylene. The addition of cysteine to reaction mixtures partially protected the enzyme system against inactivation, most notably protecting the NADH-oxidoreductase component. This suggested the participation of diffusible intermediates in the inactivation of the oxidoreductase.     

Hemoglobins dioxygenate nitric oxide with high fidelity

Distantly related members of the hemoglobin (Hb) superfamily including red blood cell Hb, muscle myoglobin (Mb) and the microbial flavohemoglobin (flavoHb) dioxygenate nitric oxide (.NO). The reaction serves important roles in .NO metabolism and detoxification throughout the aerobic biosphere. Analysis of the stoichiometric product nitrate shows greater than 99% double O-atom incorporation from Hb(18)O(2), Mb(18)O(2) and flavoHb(18)O(2) demonstrating a conserved high fidelity .NO dioxygenation mechanism. Whereas, reactions of .NO with the structurally unrelated Turbo cornutus MbO(2) or free superoxide radical (-O.(2)) yield sub-stoichiometric nitrate showing low fidelity O-atom incorporation. These and other results support a .NO dioxygenation mechanism involving (1) rapid reaction of .NO with a Fe(III-)O.(2) intermediate to form Fe(III-)OONO and (2) rapid isomerization of the Fe(III-)OONO intermediate to form nitrate. A sub-microsecond isomerization event is hypothesized in which the O-O bond homolyzes to form a protein caged [Fe(IV)O .NO(2)] intermediate and ferryl oxygen attacks .NO(2) to form nitrate. Hb functions as a .NO dioxygenase by controlling O(2) binding and electrochemistry, guiding .NO diffusion and reaction, and shielding highly reactive intermediates from solvent water and biomolecules.     

A novel toluene-3-monooxygenase pathway cloned from Pseudomonas pickettii PKO1.

Plasmid pRO1957, which contains a 26.5-kb fragment from the chromosome of Pseudomonas pickettii PKO1, allows P. aeruginosa PAO1 to grow on toluene or benzene as a sole carbon and energy source. A subclone of pRO1957, designated pRO1966, when present in P. aeruginosa PAO1 grown in lactate-toluene medium, accumulates m-cresol in the medium, indicating that m-cresol is an intermediate of toluene catabolism. Moreover, incubation of such cells in the presence of 18O2 followed by gas chromatography-mass spectrometry analysis of m-cresol extracts showed that the oxygen in m-cresol was derived from molecular oxygen. Accordingly, this suggests that toluene-3-monooxygenation is the first step in the degradative pathway. Toluene-3-monooxygenase activity is positively regulated from a locus designated tbuT. Induction of the toluene-3-monooxygenase is mediated by either toluene, benzene, ethylbenzene, or m-cresol. Moreover, toluene-3-monooxygenase activity induced by these effectors also metabolizes benzene and ethylbenzene to phenol and 3-ethylphenol, respectively, and also after induction, o-xylene, m-xylene, and p-xylene are metabolized to 3,4-dimethylphenol, 2,4-dimethylphenol, and 2,5-dimethylphenol, respectively, although the xylene substrates are not effectors. Styrene and phenylacetylene are transformed into more polar products.     

A monooxygenase catalyzes sequential dechlorinations of 2,4,6-trichlorophenol by oxidative and hydrolytic reactions

Ralstonia eutropha JMP134 2,4,6-trichlorophenol (2,4,6-TCP) 4-monooxygenase catalyzes sequential dechlorinations of 2,4,6-TCP to 6-chlorohydroxyquinol. Although 2,6-dichlorohydroxyquinol is a logical metabolic intermediate, the enzyme hardly uses it as a substrate, implying it may not be a true intermediate. Evidence is provided to support the proposition that the monooxygenase oxidized 2,4,6-TCP to 2,6-dichloroquinone that remained with the enzyme and got hydrolyzed to 2-chlorohydroxyquinone, which was chemically reduced by ascorbate and NADH to 6-chlorohydroxyquinol. When the monooxygenase oxidized 2,6-dichlorophenol, the product was 2,6-dichloroquinol, which was not further converted to 6-chlorohydroxyquinol, implying that the enzyme only converts 2,6-dichloroquinone to 6-chlorohydroxyquinol. Stoichiometric analysis indicated the consumption of one O2 molecule per 2,4,6-TCP converted to 6-chlorohydroxyquinol, ruling out the possibility of two oxidative reactions. Experiments with 18O-labeling gave direct evidence for the incorporation of oxygen from both O2 and H2O into the produced 6-chlorohydroxyquinol. A monooxygenase that catalyzes hydroxylation by both oxidative and hydrolytic reactions has not been reported to date. The ability of the enzyme to perform two types of reactions is not due to the presence of a second functional domain but rather is due to catalytic promiscuity, as a homologous monooxygenase converts 2,4,6-TCP to only 2,6-dichloroquinol. Employing both conventional catalysis and catalytic promiscuity of a single enzyme in two consecutive steps of a metabolic pathway has been unknown previously.     

Toluene-4-monooxygenase, a three-component enzyme system that catalyzes the oxidation of toluene to p-cresol in Pseudomonas mendocina KR1.

Pseudomonas mendocina KR1 grows on toluene as a sole carbon and energy source. A multicomponent oxygenase was partially purified from toluene-grown cells and separated into three protein components. The reconstituted enzyme system, in the presence of NADH and Fe2+, oxidized toluene to p-cresol as the first detectable product. Experiments with p-deutero-toluene led to the isolation of p-cresol which retained 68% of the deuterium initially present in the parent molecule. When the reconstituted enzyme system was incubated with toluene in the presence of 18O2, the oxygen in p-cresol was shown to be derived from molecular oxygen. The results demonstrate that P. mendocina KR1 initiates degradation of toluene by a multicomponent enzyme system which has been designated toluene-4-monooxygenase.     

Toluene monooxygenase-catalyzed epoxidation of alkenes

Several toluene monooxygenase-producing organisms were tested for their ability to oxidize linear alkenes and chloroalkenes three to eight carbons long. Each of the wild-type organisms degraded all of the alkenes that were tested. Epoxides were produced during the oxidation of butene, butadiene, and pentene but not hexene or octadiene. A strain of Escherichia coli expressing the cloned toluene-4-monooxygenase (T4MO) of Pseudomonas mendocina KR1 was able to oxidize butene, butadiene, pentene, and hexene but not octadiene, producing epoxides from all of the substrates that were oxidized. A T4MO-deficient variant of P. mendocina KR1 oxidized alkenes that were five to eight carbons long, but no epoxides were detected, suggesting the presence of multiple alkene-degrading enzymes in this organism. The alkene oxidation rates varied widely (ranging from 0. 01 to 0.33 micromol of substrate/min/mg of cell protein) and were specific for each organism-substrate pair. The enantiomeric purity of the epoxide products also varied widely, ranging from 54 to >90% of a single epoxide enantiomer. In the absence of more preferred substrates, such as toluene or alkenes, the epoxides underwent further toluene monooxygenase-catalyzed transformations, forming products that were not identified.     

Toluene 2-Monooxygenase-Dependent Growth of Burkholderia cepacia G4/PR1 on Diethyl Ether

Aerobic bacterial growth on aromatic hydrocarbons typically requires oxygenase enzymes, which are known to fortuitously oxidize nongrowth substrates. In this study, we found that oxidation of diethyl ether by toluene 2-monooxygenase supported more rapid growth of Burkholderia cepacia G4/PR1 than did the aromatic substrates n-propylbenzene and o-xylene. The wild-type Burkholderia cepacia G4 failed to grow on diethyl ether. Purified toluene 2-monooxygenase protein components oxidized diethyl ether stoichiometrically to ethanol and acetaldehyde. Butyl methyl ether, diethyl sulfide, and 2-chloroethyl ethyl ether were oxidized by B. cepacia G4/PR1.     

Methane monooxygenase catalyzed oxygenation of 1,1-dimethylcyclopropane. Evidence for radical and carbocationic intermediates

Methane monooxygenase catalyzes the oxygenation of 1,1-dimethylcyclopropane in the presence of O2 and NADH to (1-methylcyclopropyl)methanol (81%), 3-methyl-3-buten-1-ol (6%), and 1-methyl-cyclobutanol (13%). Oxygenation by 18O2 using the purified enzyme proceeds with incorporation of 18O into the products. Inasmuch as methane monooxygenase catalyzes the insertion of O from O2 into a carbon-hydrogen bond of alkanes, (1-methylcyclopropyl)methanol appears to be a conventional oxygenation product. 3-Methyl-3-buten-1-ol is a rearrangement product that can be rationalized on the basis that enzymatic oxygenation of 1,1-dimethylcyclopropane proceeds via the (1-methylcyclopropyl)carbinyl radical, which is expected to undergo rearrangement with ring opening to the homoallylic 3-methyl-3-buten-1-yl radical in competition with conventional oxygenation. Oxygenation of the latter radical gives 3-methyl-3-buten-1-ol. 1-Methylcyclobutanol is a ring-expansion product, whose formation is best explained on the basis that the 1-methylcyclobutyl tertiary carbocation is an oxygenation intermediate. This cation would result from rearrangements of carbocations derived by one-electron oxidation of either radical intermediate. The fact that both 3-methyl-3-buten-1-ol and 1-methylcyclobutanol are produced suggests that the oxygenation mechanism involves both radical and carbocationic intermediates. Radicals and carbocations can both be intermediates if they are connected by an electron-transfer step. A reasonable reaction sequence is one in which the cofactor (mu-oxo)diiron reacts with O2 and two electrons to generate a hydrogen atom abstracting species and an oxidizing agent. The hydrogen-abstracting species might be the enzymic radical or another species generated by the iron complex and O2.(ABSTRACT TRUNCATED AT 250 WORDS)