Protein Engineering of Toluene-o-Xylene Monooxygenase from Pseudomonas stutzeri OX1 for Synthesizing 4-Methylresorcinol, Methylhydroquinone, and Pyrogallol

Toluene-o-xylene monooxygenase (ToMO) from Pseudomonas stutzeri OX1 oxidizes toluene to 3- and 4-methylcatechol and oxidizes benzene to form phenol; in this study ToMO was found to also form catechol and 1,2,3-trihydroxybenzene (1,2,3-THB) from phenol. To synthesize novel dihydroxy and trihydroxy derivatives of benzene and toluene, DNA shuffling of the alpha-hydroxylase fragment of ToMO (TouA) and saturation mutagenesis of the TouA active site residues I100, Q141, T201, and F205 were used to generate random mutants. The mutants were initially identified by screening with a rapid agar plate assay and then were examined further by high-performance liquid chromatography and gas chromatography. Several regiospecific mutants with high rates of activity were identified; for example, Escherichia coli TG1/pBS(Kan)ToMO expressing the F205G TouA saturation mutagenesis variant formed 4-methylresorcinol (0.78 nmol/min/mg of protein), 3-methylcatechol (0.25 nmol/min/mg of protein), and methylhydroquinone (0.088 nmol/min/mg of protein) from o-cresol, whereas wild-type ToMO formed only 3-methylcatechol (1.1 nmol/min/mg of protein). From o-cresol, the I100Q saturation mutagenesis mutant and the M180T/E284G DNA shuffling mutant formed methylhydroquinone (0.50 and 0.19 nmol/min/mg of protein, respectively) and 3-methylcatechol (0.49 and 1.5 nmol/min/mg of protein, respectively). The F205G mutant formed catechol (0.52 nmol/min/mg of protein), resorcinol (0.090 nmol/min/mg of protein), and hydroquinone (0.070 nmol/min/mg of protein) from phenol, whereas wild-type ToMO formed only catechol (1.5 nmol/min/mg of protein). Both the I100Q mutant and the M180T/E284G mutant formed hydroquinone (1.2 and 0.040 nmol/min/mg of protein, respectively) and catechol (0.28 and 2.0 nmol/min/mg of protein, respectively) from phenol. Dihydroxybenzenes were further oxidized to trihydroxybenzenes with different regiospecificities; for example, the I100Q mutant formed 1,2,4-THB from catechol, whereas wild-type ToMO formed 1,2,3-THB (pyrogallol). Regiospecific oxidation of the natural substrate toluene was also checked; for example, the I100Q mutant formed 22% o-cresol, 44% m-cresol, and 34% p-cresol, whereas wild-type ToMO formed 32% o-cresol, 21% m-cresol, and 47% p-cresol.     

Saturation mutagenesis of Bradyrhizobium sp. BTAi1 toluene 4-monooxygenase at alpha-subunit residues proline 101, proline 103, and histidine 214 for regiospecific oxidation of aromatics

A novel toluene monooxygenase (TMO) six-gene cluster from Bradyrhizobium sp. BTAi1 having an overall 35, 36, and 38 % protein similarity with toluene o-xylene monooxygenase (ToMO) of Pseudomonas sp. OX1, toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1, and toluene-para-monooxygenase (TpMO) of Ralstonia pickettii PKO1, respectively, was cloned and expressed in Escherichia coli TG1, and its potential activity was investigated for aromatic hydroxylation and trichloroethylene (TCE) degradation. The natural substrate toluene was hydroxylated to p-cresol, indicating that the new toluene monooxygenase (T4MO·BTAi1) acts as a para hydroxylating enzyme, similar to T4MO and TpMO. Some shifts in regiospecific hydroxylations were observed compared to the other wild-type TMOs. For example, wild-type T4MO·BTAi1 formed catechol (88 %) and hydroquinone (12 %) from phenol, whereas all the other wild-type TMOs were reported to form only catechol. Furthermore, it was discovered that TG1 cells expressing wild-type T4MO·BTAi1 mineralized TCE at a rate of 0.67?±?0.10 nmol Cl^?/h/mg protein. Saturation and site directed mutagenesis were used to generate eight variants of T4MO·BTAi1 at alpha-subunit positions P101, P103, and H214: P101T/P103A, P101S, P101N/P103T, P101V, P103T, P101V/P103T, H214G, and H214G/D278N; by testing the substrates phenol, nitrobenzene, and naphthalene, positions P101 and P103 were found to influence the regiospecific oxidation of aromatics. For example, compared to wild type, variant P103T produced four fold more m-nitrophenol from nitrobenzene as well as produced mainly resorcinol (60 %) from phenol whereas wild-type T4MO·BTAi1 did not. Similarly, variants P101T/P103A and P101S synthesized more 2-naphthol and 2.3-fold and 1.6-fold less 1-naphthol from naphthalene, respectively.     

Alanine 101 and alanine 110 of the alpha subunit of Pseudomonas stutzeri OX1 toluene-o-xylene monooxygenase influence the regiospecific oxidation of aromatics

Saturation mutagenesis was used to generate 10 mutants of toluene-o-xylene monooxygenase (ToMO) at alpha subunit (TouA) positions A101 and A110: A101G, A101I, A101M, A101VE, A101V, A110G, A110C, A110S, A110P, and A110T; by testing the substrates toluene, o-cresol, m-cresol, p-cresol, phenol, naphthalene, o-methoxyphenol, m-methoxyphenol, p-methoxyphenol, o-xylene, and nitrobenzene, these positions were found to influence the regiospecific oxidation of aromatics. For example, compared to wild-type ToMO, TouA variant A101V produced threefold more 3-methoxycatechol from m-methoxyphenol as well as produced methylhydroquinone from o-cresol whereas wild-type ToMO did not. Similarly, variant A110C synthesized 1.8-fold more o-cresol from toluene and 1.8-fold more 3-methoxycatechol from m-methoxyphenol, and variant A110G synthesized more m-nitrophenol and twofold less p-nitrophenol from nitrobenzene. The A101V and A110C mutations did not affect the rate of reaction with the natural substrate toluene, so the variants had high activity. This is the first report that these or analogous residues influence the catalysis with this class of enzymes. Wild-type ToMO was found to oxidize o-methoxyphenol to methoxyhydroquinone (60%) and 4-methoxyresorcinol (40%), m-methoxyphenol to 4-methoxycatechol (96%) and 3-methoxycatechol (4%), and p-methoxyphenol to 4-methoxycatechol (100%).     

Protein engineering of toluene-o-xylene monooxygenase from Pseudomonas stutzeri OX1 for synthesizing 4-methylresorcinol, methylhydroquinone, and pyrogallol

Toluene-o-xylene monooxygenase (ToMO) from Pseudomonas stutzeri OX1 oxidizes toluene to 3- and 4-methylcatechol and oxidizes benzene to form phenol; in this study ToMO was found to also form catechol and 1,2,3-trihydroxybenzene (1,2,3-THB) from phenol. To synthesize novel dihydroxy and trihydroxy derivatives of benzene and toluene, DNA shuffling of the alpha-hydroxylase fragment of ToMO (TouA) and saturation mutagenesis of the TouA active site residues I100, Q141, T201, and F205 were used to generate random mutants. The mutants were initially identified by screening with a rapid agar plate assay and then were examined further by high-performance liquid chromatography and gas chromatography. Several regiospecific mutants with high rates of activity were identified; for example, Escherichia coli TG1/pBS(Kan)ToMO expressing the F205G TouA saturation mutagenesis variant formed 4-methylresorcinol (0.78 nmol/min/mg of protein), 3-methylcatechol (0.25 nmol/min/mg of protein), and methylhydroquinone (0.088 nmol/min/mg of protein) from o-cresol, whereas wild-type ToMO formed only 3-methylcatechol (1.1 nmol/min/mg of protein). From o-cresol, the I100Q saturation mutagenesis mutant and the M180T/E284G DNA shuffling mutant formed methylhydroquinone (0.50 and 0.19 nmol/min/mg of protein, respectively) and 3-methylcatechol (0.49 and 1.5 nmol/min/mg of protein, respectively). The F205G mutant formed catechol (0.52 nmol/min/mg of protein), resorcinol (0.090 nmol/min/mg of protein), and hydroquinone (0.070 nmol/min/mg of protein) from phenol, whereas wild-type ToMO formed only catechol (1.5 nmol/min/mg of protein). Both the I100Q mutant and the M180T/E284G mutant formed hydroquinone (1.2 and 0.040 nmol/min/mg of protein, respectively) and catechol (0.28 and 2.0 nmol/min/mg of protein, respectively) from phenol. Dihydroxybenzenes were further oxidized to trihydroxybenzenes with different regiospecificities; for example, the I100Q mutant formed 1,2,4-THB from catechol, whereas wild-type ToMO formed 1,2,3-THB (pyrogallol). Regiospecific oxidation of the natural substrate toluene was also checked; for example, the I100Q mutant formed 22% o-cresol, 44% m-cresol, and 34% p-cresol, whereas wild-type ToMO formed 32% o-cresol, 21% m-cresol, and 47% p-cresol.     

The roles of intermediates in biodegradation of benzene,toluene, and p-xylene by Pseudomonas putida F1

Several types of biodegradation experiments with benzene, toluene, or p-xylene show accumulation of intermediates by Pseudomonas putida F1. Under aerobic conditions, the major intermediates identified for benzene, toluene, and p-xylene are catechol, 3-methylcatechol, and 3,6-dimethylcatechol, respectively. Oxidations of catechol and 3-methylcatechol are linked to biomass synthesis. When oxygen is limited in the system, phenol (from benzene) and m-cresol and o-cresol (from toluene) accumulate.     

Physiological relevance of successive hydroxylations of toluene by toluene para-monooxygenase of Ralstonia pickettii PKO1

We have recently found that toluene para-monooxygenase (TpMO) of Ralstonia pickettii PKO1 (encoded by tbuA1UBVA2C) performs successive hydroxylations of benzene (Appl. Environ. Microbiol. 70: 3814, 2004) as well as hydroxylates toluene to a mixture of 90% p-cresol and 10% m-cresol which are then further oxidized to 100% 4-methylcatechol (J. Bacteriol. 186: 3117, 2004) whereas it was thought previously that TpMO forms 100% m-cresol and is not capable of successive hydroxylations. Here we propose a modification of the degradation pathway originally described by Olsen et al. (J. Bacteriol. 176: 3749, 1994) that now relies primarily on TpMO for conversion of toluene to 4-methylcatechol (instead of m-cresol) since both m-cresol and p-cresol are shown here to be good substrates for Escherichia coli expressing TpMO (V_max/K_m=0.046, 0.036, and 0.055 mL min^?1 mg^?1 protein for the oxidation of toluene, m-cresol, and p-cresol, respectively). In light of the broader activity of TpMO, phenol hydroxylase (encoded by tbuD) appears to facilitate conversion of any m-cresol or p-cresol formed from toluene oxidation by TpMO to 4-methylcatechol; hence, the cell has a redundant method for making this important intermediate 4-methylcatechol. Further, it is suggested that the physiological relevance of the 10% m-cresol formed from toluene oxidation by TpMO is needed for induction of the meta cleavage operon tbuWEFGKIHJ to enable full metabolism of toluene since p-cresol (and o-cresol) do not induce the meta-cleavage pathway. Therefore both the successive hydroxylation of toluene by TpMO and the product distribution are of physiological relevance to the cell.     

Novel Pathway of Toluene Catabolism in the Trichloroethylene-Degrading Bacterium G4

o-Cresol and 3-methylcatechol were identified as successive transitory intermediates of toluene catabolism by the trichloroethylene-degrading bacterium G4. The absence of a toluene dihydrodiol intermediate or toluene dioxygenase and toluene dihydrodiol dehydrogenase activities suggested that G4 catabolizes toluene by a unique pathway. Formation of a hybrid species of ¹⁸O- and ¹⁶O-labeled 3-methylcatechol from toluene in an atmosphere of ¹⁸O₂ and ¹⁶O₂ established that G4 catabolizes toluene by successive monooxygenations at the ortho and meta positions. Detection of trace amounts of 4-methylcatechol from toluene catabolism suggested that the initial hydroxylation of toluene was not exclusively at the ortho position. Further catabolism of 3-methylcatechol was found to proceed via catechol-2,3-dioxygenase and hydroxymuconic semialdehyde hydrolase activities.     

Mutation of Glutamic Acid 103 of Toluene o-Xylene Monooxygenase as a Means To Control the Catabolic Efficiency of a Recombinant Upper Pathway for Degradation of Methylated Aromatic Compounds

Toluene o-xylene monooxygenase (ToMO) and phenol hydroxylase (PH) of Pseudomonas stutzeri OX1 act sequentially in a recombinant upper pathway for the degradation of aromatic hydrocarbons. The catalytic efficiency and regioselectivity of these enzymes optimize the degradation of growth substrates like toluene and o-xylene. For example, the sequential monooxygenation of o-xylene by ToMO and PH leads to almost exclusive production of 3,4-dimethylcatechol (3,4-DMC), the only isomer that can be further metabolized by the P. stutzeri meta pathway. We investigated the possibility of producing ToMO mutants with modified regioselectivity compared with the regioselectivity of the wild-type protein in order to alter the ability of the recombinant upper pathway to produce methylcatechol isomers from toluene and to produce 3,4-DMC from o-xylene. The combination of mutant (E103G)-ToMO and PH increased the production of 4-methylcatechol from toluene and increased the formation of 3,4-DMC from o-xylene. These data strongly support the idea that the products and efficiency of the metabolic pathway can be controlled not only through mutations that increase the catalytic efficiency of the enzymes involved but also through tuning the substrate specificity and regioselectivity of the enzymes. These findings are crucial for the development of future metabolic engineering strategies.     

Regiospecific oxidation of naphthalene and fluorene by toluene monooxygenases and engineered toluene 4-monooxygenases of Pseudomonas mendocina KR1

The regiospecific oxidation of the polycyclic aromatic hydrocarbons naphthalene and fluorene was examined with Escherichia coli strains expressing wildtype toluene 4-monooxygenase (T4MO) from Pseudomonas mendocina KR1, toluene para-monooxygenase (TpMO) from Ralstonia pickettii PKO1, toluene ortho-monooxygenase (TOM) from Burkholderia cepacia G4, and toluene/ortho-xylene monooxygenase (ToMO) from P. stutzeri OX1. T4MO oxidized toluene (12.1+/-0.8 nmol/min/mg protein at 109 microM), naphthalene (7.7+/-1.5 nmol/min/mg protein at 5 mM), and fluorene (0.68+/-0.04 nmol/min/mg protein at 0.2 mM) faster than the other wildtype enzymes (2-22-fold) and produced a mixture of 1-naphthol (52%) and 2-naphthol (48%) from naphthalene, which was successively transformed to a mixture of 2,3-, 2,7-, 1,7-, and 2,6-dihydroxynaphthalenes (7%, 10%, 20%, and 63%, respectively). TOM and ToMO made 1,7-dihydroxynaphthalene from 1-naphthol, and ToMO made a mixture of 2,3-, 2,6-, 2,7-, and 1,7-dihydroxynaphthalene (26%, 22%, 1%, and 44%, respectively) from 2-naphthol. TOM had no activity on 2-naphthol, and T4MO had no activity on 1-naphthol. To take advantage of the high activity of wildtype T4MO but to increase its regiospecificity on naphthalene, seven engineered enzymes containing mutations in T4MO alpha hydroxylase TmoA were examined; the selectivity for 2-naphthol by T4MO I100A, I100S, and I100G was enhanced to 88-95%, and the selectivity for 1-naphthol was enhanced to 87% and 99% by T4MO I100L and G103S/A107G, respectively, while high oxidation rates were maintained except for G103S/A107G. Therefore, the regiospecificity for naphthalene oxidation was altered to practically pure 1-naphthol or 2-naphthol. All four wildtype monooxygenases were able to oxidize fluorene to different monohydroxylated products; T4MO oxidized fluorene successively to 3-hydroxyfluorene and 3,6-dihydroxyfluorene, which was confirmed by gas chromatography-mass spectrometry and 1H nuclear magnetic resonance analysis. TOM and its variant TomA3 V106A oxidize fluorene to a mixture of 1-, 2-, 3-, and 4-hydroxyfluorene. This is the first report of using enzymes to synthesize 1-, 3-, and 4-hydroxyfluorene, and 3,6-dihydroxyfluorene from fluorene as well as 2-naphthol and 2,6-dihydroxynaphthalene from naphthalene.     

Mutations of Toluene-4-Monooxygenase That Alter Regiospecificity of Indole Oxidation and Lead to Production of Novel Indigoid Pigments

Broad-substrate-range monooygenase enzymes, including toluene-4-monooxygenase (T4MO), can catalyze the oxidation of indole. The indole oxidation products can then condense to form the industrially important dye indigo. Site-directed mutagenesis of T4MO resulted in the creation of T4MO isoforms with altered pigment production phenotypes. High-pressure liquid chromatography, thin-layer chromatography, and nuclear magnetic resonance analysis of the indole oxidation products generated by the mutant T4MO isoforms revealed that the phenotypic differences were primarily due to changes in the regiospecificity of indole oxidation. Most of the mutations described in this study changed the ratio of the primary indole oxidation products formed (indoxyl, 2-oxindole, and isatin), but some mutations, particularly those involving amino acid G103 of tmoA, allowed for the formation of additional products, including 7-hydroxyindole and novel indigoid pigments. For example, mutant G103L converted 17% of added indole to 7-hydroxyindole and 29% to indigoid pigments including indigo and indirubin and two other structurally related pigments. The double mutant G103L:A107G converted 47% of indole to 7-hydroxyindole, but no detectable indigoid pigments were formed, similar to the product distribution observed with the toluene-2-monooxygenase (T2MO) of Burkholderia cepacia G4. These results demonstrate that modification of the tmoA active site can change the products produced by the enzyme and lead to the production of novel pigments and other indole oxidation products with potential commercial and medicinal utility.     

Regiospecificity of Two Multicomponent Monooxygenases from Pseudomonas stutzeri OX1: Molecular Basis for Catabolic Adaptation of This Microorganism to Methylated Aromatic Compounds

The pathways for degradation of aromatic hydrocarbons are constantly modified by a variety of genetic mechanisms. Genetic studies carried out with Pseudomonas stutzeri OX1 suggested that the tou operon coding for toluene o-xylene monooxygenase (ToMO) was recently recruited into a preexisting pathway that already possessed the ph operon coding for phenol hydroxylase (PH). This apparently resulted in a redundancy of enzymatic activities, because both enzymes are able to hydroxylate (methyl)benzenes to (methyl)catechols via the intermediate production of (methyl)phenols. We investigated the kinetics and regioselectivity of toluene and o-xylene oxidation using Escherichia coli cells expressing ToMO and PH complexes. Our data indicate that in the recombinant system the enzymes act sequentially and that their catalytic efficiency and regioselectivity optimize the degradation of toluene and o-xylene, both of which are growth substrates. The main product of toluene oxidation by ToMO is p-cresol, the best substrate for PH, which catalyzes its transformation to 4-methylcatechol. The sequential action of the two enzymes on o-xylene leads, via the intermediate 3,4-dimethylphenol, to the exclusive production of 3,4-dimethylcatechol, the only dimethylcatechol isomer that can serve as a carbon and energy source after further metabolic processing. Moreover, our data strongly support a metabolic explanation for the acquisition of the ToMO operon by P. stutzeri OX1. It is possible that using the two enzymes in a concerted fashion confers on the strain a selective advantage based on the ability of the microorganism to optimize the efficiency of the use of nonhydroxylated aromatic hydrocarbons, such as benzene, toluene, and o-xylene.     

New metabolic pathway for o-cresol degradation by Pseudomonas sp. CP4 as evidenced by 1H NMR spectroscopic studies

Degradation intermediates of o-, m- and p-cresols extracted from resting cells of Pseudomonas sp. CP4, a potent cresol- and phenol-degrading laboratory isolate, were analysed by using ¹H NMR spectroscopy at 270 MHz. Ortho-, meta- and para-cresols were found to be degraded to 2-methyl-4-oxalocrotonate. 3-Methylcatechol from o-cresol was degraded further to 2-ketohex-cis-4-enoate, 4-methylcatechol from m- and p-cresol was degraded to 2-ketohex-cis-4-enoate. Also 2-ketopent-4-enoate was found to be formed from p-cresol. Formation of 2-methyl-4-oxalocrotonate was envisaged as taking place from 5-hydroxy-2-methylmuconic semialdehyde, the ring-cleavage product of 4-methylresorcinol, a possible product by hydroxylation of o-cresol along with 3-methylcatechol. This is a deviation from the hitherto known pathways of o-cresol degradation. Based on these observations, pathways for the degradation of all three isomers of cresol are proposed.     

Toluene 3-monooxygenase of Ralstonia pickettii PKO1 is a para-hydroxylating enzyme

Oxygenases are promising biocatalysts for performing selective hydroxylations not accessible by chemical methods. Whereas toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1 hydroxylates monosubstituted benzenes at the para position and toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 hydroxylates at the ortho position, toluene 3-monooxygenase (T3MO) of Ralstonia pickettii PKO1 was reported previously to hydroxylate toluene at the meta position, producing primarily m-cresol (R. H. Olsen, J. J. Kukor, and B. Kaphammer, J. Bacteriol. 176:3749-3756, 1994). Using gas chromatography, we have discovered that T3MO hydroxylates monosubstituted benzenes predominantly at the para position. TG1/pBS(Kan)T3MO cells expressing T3MO oxidized toluene at a maximal rate of 11.5 +/- 0.33 nmol/min/mg of protein with an apparent Km value of 250 microM and produced 90% p-cresol and 10% m-cresol. This product mixture was successively transformed to 4-methylcatechol. T4MO, in comparison, produces 97% p-cresol and 3% m-cresol. Pseudomonas aeruginosa PAO1 harboring pRO1966 (the original T3MO-bearing plasmid) also exhibited the same product distribution as that of TG1/pBS(Kan)T3MO. TG1/pBS(Kan)T3MO produced 66% p-nitrophenol and 34% m-nitrophenol from nitrobenzene and 100% p-methoxyphenol from methoxybenzene, as well as 62% 1-naphthol and 38% 2-naphthol from naphthalene; similar results were found with TG1/pBS(Kan)T4MO. Sequencing of the tbu locus from pBS(Kan)T3MO and pRO1966 revealed complete identity between the two, thus eliminating any possible cloning errors. 1H nuclear magnetic resonance analysis confirmed the structural identity of p-cresol in samples containing the product of hydroxylation of toluene by pBS(Kan)T3MO.     

Integrated biooxidation and acid dehydration process for monohydroxylation of aromatics

We examined the performance of an integrated biooxidation and acid dehydration process for aromatic monohydroxylation using the production of o-cresol from toluene as the model conversion. A toluene cis-glycol dehydrogenase gene (todD)-disrupted mutant of Pseudomonas putida T-57 was employed as the whole cell biocatalyst for oxidizing toluene to toluene cis-glycol (TCG). After bioconversion of toluene to TCG, o-cresol was produced by adding HCl to the culture medium, since it was found that TCG became unstable at a low pH and underwent spontaneous dehydration. When the todD-disrupted mutant cells were grown in a two-liquid-phase culture system with oleyl alcohol as the organic solvent, this integrated process produced 40 g l⁻¹ of o-cresol in the organic solvent phase at 30 h. The total concentration of o-cresol in the two-liquid-phase culture reached 6.6 g l⁻¹ at 30 h, which was approximately four-fold greater than that in the single-liquid-phase culture.     

Oxidation of Benzene to Phenol, Catechol, and 1,2,3-Trihydroxybenzene by Toluene 4-Monooxygenase of Pseudomonas mendocina KR1 and Toluene 3-Monooxygenase of Ralstonia pickettii PKO1

Aromatic hydroxylations are important bacterial metabolic processes but are difficult to perform using traditional chemical synthesis, so to use a biological catalyst to convert the priority pollutant benzene into industrially relevant intermediates, benzene oxidation was investigated. It was discovered that toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1, toluene 3-monooxygenase (T3MO) of Ralstonia pickettii PKO1, and toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 convert benzene to phenol, catechol, and 1,2,3-trihydroxybenzene by successive hydroxylations. At a concentration of 165 μM and under the control of a constitutive lac promoter, Escherichia coli TG1/pBS(Kan)T4MO expressing T4MO formed phenol from benzene at 19 ± 1.6 nmol/min/mg of protein, catechol from phenol at 13.6 ± 0.3 nmol/min/mg of protein, and 1,2,3-trihydroxybenzene from catechol at 2.5 ± 0.5nmol/min/mg of protein. The catechol and 1,2,3-trihydroxybenzene products were identified by both high-pressure liquid chromatography and mass spectrometry. When analogous plasmid constructs were used, E. coli TG1/pBS(Kan)T3MO expressing T3MO formed phenol, catechol, and 1,2,3-trihydroxybenzene at rates of 3 ± 1, 3.1 ± 0.3, and 0.26 ± 0.09 nmol/min/mg of protein, respectively, and E. coli TG1/pBS(Kan)TOM expressing TOM formed 1,2,3-trihydroxybenzene at a rate of 1.7 ± 0.3 nmol/min/mg of protein (phenol and catechol formation rates were 0.89 ± 0.07 and 1.5 ± 0.3 nmol/min/mg of protein, respectively). Hence, the rates of synthesis of catechol by both T3MO and T4MO and the 1,2,3-trihydroxybenzene formation rate by TOM were found to be comparable to the rates of oxidation of the natural substrate toluene for these enzymes (10.0 ± 0.8, 4.0 ± 0.6, and 2.4 ± 0.3 nmol/min/mg of protein for T4MO, T3MO, and TOM, respectively, at a toluene concentration of 165 μM).     

Saturation mutagenesis of Burkholderia cepacia R34 2,4-dinitrotoluene dioxygenase at DntAc valine 350 for synthesizing nitrohydroquinone, methylhydroquinone, and methoxyhydroquinone

Saturation mutagenesis of the 2,4-dinitrotoluene dioxygenase (DDO) of Burkholderia cepacia R34 at position valine 350 of the DntAc alpha-subunit generated mutant V350F with significantly increased activity towards o-nitrophenol (47 times), m-nitrophenol (34 times), and o-methoxyphenol (174 times) as well as an expanded substrate range that now includes m-methoxyphenol, o-cresol, and m-cresol (wild-type DDO had no detectable activity for these substrates). Another mutant, V350M, also displays increased activity towards o-nitrophenol (20 times) and o-methoxyphenol (162 times) as well as novel activity towards o-cresol. Products were synthesized using whole Escherichia coli TG1 cells expressing the recombinant R34 dntA loci from pBS(Kan)R34, and the initial rates of product formation were determined at 1 mM substrate by reverse-phase high-pressure liquid chromatography. V350F produced both nitrohydroquinone at a rate of 0.75 +/- 0.15 nmol/min/mg of protein and 3-nitrocatechol at a rate of 0.069 +/- 0.001 nmol/min/mg of protein from o-nitrophenol, 4-nitrocatechol from m-nitrophenol at 0.29 +/- 0.02 nmol/min/mg of protein, methoxyhydroquinone from o-methoxyphenol at 2.5 +/- 0.6 nmol/min/mg of protein, methoxyhydroquinone from m-methoxyphenol at 0.55 +/- 0.02 nmol/min/mg of protein, both methylhydroquinone at 1.52 +/- 0.02 nmol/min/mg of protein and 2-hydroxybenzyl alcohol at 0.74 +/- 0.05 nmol/min/mg of protein from o-cresol, and methylhydroquinone at 0.43 +/- 0.1 nmol/min/mg of protein from m-cresol. V350M produced both nitrohydroquinone at a rate of 0.33 nmol/min/mg of protein and 3-nitrocatechol at 0.089 nmol/min/mg of protein from o-nitrophenol, methoxyhydroquinone from o-methoxyphenol at 2.4 nmol/min/mg of protein, methylhydroquinone at 1.97 nmol/min/mg of protein and 2-hydroxybenzyl alcohol at 0.11 nmol/min/mg of protein from o-cresol. The DDO variants V350F and V350M also exhibited 10-fold-enhanced activity towards naphthalene (8 +/- 2.6 nmol/min/mg of protein), forming (1R,2S)-cis-1,2-dihydro-1,2-dihydroxynaphthalene. Hence, mutagenesis of wild-type DDO through active-site engineering generated variants with relatively high rates toward a previously uncharacterized class of substituted phenols for the nitroarene dioxygenases; seven previously uncharacterized substrates were evaluated for wild-type DDO, and four novel monooxygenase-like products were found for the DDO variants V350F and V350M (methoxyhydroquinone, methylhydroquinone, 2-hydroxybenzyl alcohol, and 3-nitrocatechol).     

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.     

Biotransformation of benzene and toluene to catechols by phenol hydroxylase from Arthrobacter sp. W1

Phenol hydroxylase gene engineered microorganism (PH_IND) was used to synthesize catechols from benzene and toluene by successive hydroxylation reaction. HPLC-MS and ¹H NMR analysis proved that the products of biotransformation were the corresponding catechols via the intermediate production of phenols. It was indicated that the main products of toluene oxidation were o-cresol and p-cresol. 3-Methylcatechol was the predominant product for m-cresol biotransformation. Formation rate of catechol (25 μM/min/g cell dry weight) was 1.43-fold higher than that of methylcatechols. It was suggested that phenol hydroxylase could be successfully used to transform both benzene and toluene to catechols by successive hydroxylation.     

Monocyclic Aromatic Hydrocarbon Degradation by Rhodococcus sp. Strain DK17

Rhodococcus sp. strain DK17 was isolated from soil and analyzed for the ability to grow on o-xylene as the sole carbon and energy source. Although DK17 cannot grow on m- and p-xylene, it is capable of growth on benzene, phenol, toluene, ethylbenzene, isopropylbenzene, and other alkylbenzene isomers. One UV-generated mutant strain, DK176, simultaneously lost the ability to grow on o-xylene, ethylbenzene, isopropylbenzene, toluene, and benzene, although it could still grow on phenol. The mutant strain was also unable to oxidize indole to indigo following growth in the presence of o-xylene. This observation suggests the loss of an oxygenase that is involved in the initial oxidation of the (alkyl)benzenes tested. Another mutant strain, DK180, isolated for the inability to grow on o-xylene, retained the ability to grow on benzene but was unable to grow on alkylbenzenes due to loss of a meta-cleavage dioxygenase needed for metabolism of methyl-substituted catechols. Further experiments showed that DK180 as well as the wild-type strain DK17 have an ortho-cleavage pathway which is specifically induced by benzene but not by o-xylene. These results indicate that DK17 possesses two different ring-cleavage pathways for the degradation of aromatic compounds, although the initial oxidation reactions may be catalyzed by a common oxygenase. Gas chromatography-mass spectrometry and 300-MHz proton nuclear magnetic resonance spectrometry clearly show that DK180 accumulates 3,4-dimethylcatechol from o-xylene and both 3- and 4-methylcatechol from toluene. This means that there are two initial routes of oxidation of toluene by the strain. Pulsed-field gel electrophoresis analysis demonstrated the presence of two large megaplasmids in the wild-type strain DK17, one of which (pDK2) was lost in the mutant strain DK176. Since several other independently derived mutant strains unable to grow on alkylbenzenes are also missing pDK2, the genes encoding the initial steps in alkylbenzene metabolism (but not phenol metabolism) appear to be present on this approximately 330-kb plasmid.     

Protein engineering of toluene-o-xylene monooxygenase from Pseudomonas stutzeri OX1 for enhanced chlorinated ethene degradation and o-xylene oxidation

Toluene-o-xylene monooxygenase (ToMO) from Pseudomonas stutzeri OX1 has been shown to degrade all chlorinated ethenes individually and as mixtures. Here, DNA shuffling of the alpha hydroxylase fragment of ToMO (TouA) and saturation mutagenesis of the TouA active site residues I100, Q141, T201, F205, and E214 were used to enhance the degradation of chlorinated aliphatics. The ToMO mutants were identified using a chloride ion screen and then were further examined by gas chromatography. Escherichia coli TG1/pBS(Kan)ToMO expressing TouA saturation mutagenesis variant I100Q was identified that has 2.8-fold better trichloroethylene (TCE) degradation activity (apparent V _max of 1.77 nmol min⁻¹ mg⁻¹ protein⁻¹ vs 0.63 nmol min⁻¹ mg⁻¹ protein⁻¹). Another variant, E214G/D312N/M399V, has 2.5-fold better cis-1,2-dichloroethylene (cis-DCE) degradation activity (apparent V _max of 8.4 nmol min⁻¹ mg⁻¹ protein⁻¹ vs 3.3 nmol min⁻¹ mg⁻¹ protein⁻¹). Additionally, the hydroxylation regiospecificity of o-xylene and naphthalene were altered significantly for ToMO variants A107T/E214A, T201G, and T201S. Variant T201S produced 2.0-fold more 2,3-dimethylphenol (2,3-DMP) from o-xylene than the wild-type ToMO, whereas variant A107T/E214A had 6.0-fold altered regiospecificity for 2,3-DMP formation. Variant A107T/E214A also produced 3.0-fold more 2-naphthol from naphthalene than the wild-type ToMO, whereas the regiospecificity of variant T201S was altered to synthesize 3.0-fold less 2-naphthol, so that it made almost exclusively 1-naphthol (96%). Variant T201G was more regiospecific than variants A107T/E214A and T201S and produced 100% 3,4-DMP from o-xylene and >99% 1-naphthol from naphthalene. Hence, ToMO activity was enhanced for the degradation of TCE and cis-DCE and for the regiospecific hydroxylation of o-xylene and naphthalene through DNA shuffling and saturation mutagenesis.