Engineered marine Antarctic bacterium Pseudoalteromonas haloplanktis TAC125: a promising micro-organism for the bioremediation of aromatic compounds

Aims:  The recombinant Antarctic Pseudoalteromonas haloplanktis TAC125 ( P. haloplanktis TAC/ tou ) expressing toluene- o- xylene monooxygenase (ToMO) can efficiently convert several aromatic compounds into their corresponding catechols in a broad range of temperature. When the genome of P. haloplanktis TAC125 was analysed in silico , the presence of a DNA sequence coding for a putative laccase-like protein was revealed. It is well known that bacterial laccases are able to oxidize dioxygenated aromatic compounds such as catechols.
Methods and Results:  We analysed the catabolic features, conferred by recombinant ToMO activity and the endogenous laccase enzymatic activity, of P. haloplanktis TAC/ tou engineered strain and its ability to grow on aromatic compounds as sole carbon and energy sources.
Conclusions:  Results presented highlight the broad potentiality of P. haloplanktis TAC/ tou cells expressing recombinant ToMO in bioremediation and suggest the use of this engineered Antarctic bacterium in the bioremediation of chemically contaminated marine environments and/or cold effluents.
Significance and Impact of the Study:  This paper demonstrates the possibility to confer new and specific degradative capabilities to a bacterium isolated from an unpolluted environment (Antarctic seawater) transforming it into a bacterium able to grow on phenol as sole carbon and energy source.     

Engineering of a psychrophilic bacterium for the bioremediation of aromatic compounds

Microbial degradation of aromatic hydrocarbons has been studied with the aim of developing applications for the removal of toxic compounds. Efforts have been directed toward the genetic manipulation of mesophilic bacteria to improve their ability to degrade pollutants, even though many pollution problems occur in sea waters and in effluents of industrial processes which are characterized by low temperatures. From these considerations the idea of engineering a psychrophilic microorganism for the oxidation of aromatic compounds was developed.In a previous paper it was demonstrated that the recombinant Antarctic Pseudoalteromonas haloplanktis TAC125 (PhTAC/tou) expressing a toluene-o-xylene monooxygenase (ToMO) is able to convert several aromatic compounds into corresponding catechols. In our work we improved the metabolic capability of PhTAC/tou cells by combining action of recombinant ToMO enzyme with that of the endogenous P. haloplanktis TAC125 laccase-like protein. This strategy allowed conferring new and specific degradative capabilities to a bacterium isolated from an unpolluted environment; indeed engineered PhTAC/tou cells are able to grow on aromatic compounds as sole carbon and energy sources. Our approach demonstrates the possibility to use the engineered psychrophilic bacterium for the bioremediation of chemically contaminated marine environments and/or cold effluents.     

Tetrachloroethylene, trichloroethylene, and chlorinated phenols induce toluene-o-xylene monooxygenase activity in Pseudomonas stutzeri OX1

Pseudomonas stutzeri OX1 naphthalene-oxidation activity is induced 3.0-fold by tetrachloroethylene (PCE) and 3.1-fold by trichloroethylene (TCE) at 100 microM. With the mutant P. stutzeri M1, which does not express toluene-o-xylene monooxygenase (ToMO, product of the tou operon), no naphthalene-oxidation activity induction by PCE and TCE was found; hence, PCE and TCE induce ToMO of P. stutzeri OX1. The chlorinated phenols 2-, 3-, and 4-chlorophenol induced ToMO expression 0.58-, 0.23- and 0.37-fold, respectively, compared to the direct inducer of the pathway, o-cresol. Using P. putida PaW340 (pPP4062, pFP3028), which has the tou promoter fused to the reporter catechol-2,3-dioxygenase, and the regulator gene touR, it was determined that the tou promoter was induced directly 5.7-, 7.1-, and 5.1-fold for 2-, 3-, and 4-chlorophenol, respectively (compared to an 8.8-fold induction with o-cresol). In addition, it was found that TCE and PCE do not directly induce the tou pathway and that components other than the tou structural and regulatory genes are necessary for induction. Gas chromatography results also showed that 100 microM TCE induced its own degradation (8-9%) in 16 h in P. stutzeri OX1, and all of the stoichiometric chloride from the degraded TCE was detected in solution.     

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.     

Tuning the specificity of the recombinant multicomponent toluene o-xylene monooxygenase from Pseudomonas sp. strain OX1 for the biosynthesis of tyrosol from 2-phenylethanol

Biocatalysis is today a standard technology for the industrial production of several chemicals, and the number of biotransformation processes running on a commercial scale is constantly increasing. Among biocatalysts, bacterial multicomponent monooxygenases (BMMs), a diverse group of nonheme diiron enzymes that activate dioxygen, are of primary interest due to their ability to catalyze a variety of complex oxidations, including reactions of mono- and dihydroxylation of phenolic compounds. In recent years, both directed evolution and rational design have been successfully used to identify the molecular determinants responsible for BMM regioselectivity and to improve their activity toward natural and nonnatural substrates. Toluene o-xylene monooxygenase (ToMO) is a BMM isolated from Pseudomonas sp. strain OX1 which hydroxylates a wide spectrum of aromatic compounds. In this work we investigate the use of recombinant ToMO for the biosynthesis in recombinant cells of Escherichia coli strain JM109 of 4-hydroxyphenylethanol (tyrosol), an antioxidant present in olive oil, from 2-phenylethanol, a cheap and commercially available substrate. We initially found that wild-type ToMO is unable to convert 2-phenylethanol to tyrosol. This was explained by using a computational model which analyzed the interactions between ToMO active-site residues and the substrate. We found that residue F176 is the major steric hindrance for the correct positioning of the reaction intermediate leading to tyrosol production into the active site of the enzyme. Several mutants were designed and prepared, and we found that the combination of different mutations at position F176 with mutation E103G allows ToMO to convert up to 50% of 2-phenylethanol into tyrosol in 2 h.     

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.     

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.     

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.     

Protein engineering of toluene-o-xylene monooxygenase from Pseudomonas stutzeri OX1 for oxidizing nitrobenzene to 3-nitrocatechol, 4-nitrocatechol, and nitrohydroquinone

Toluene-o-xylene monooxygenase (ToMO) from Pseudomonas stutzeri OX1 was found to oxidize nitrobenzene (NB) to form m-nitrophenol (m-NP, 72%) and p-NP (28%) with an initial rate of 0.098 and 0.031 nmol/(min mg protein), respectively. It was also discovered that wild-type ToMO forms 4-nitrocatechol (4-NC) from m-NP and p-NP with an initial rate of 0.15 and 0.0082 nmol/(min mg protein), respectively, and 3-NC (12%) and nitrohydroquinone (NHQ, 88%) from o-NP with an initial rate of 0.11 and 0.8 nmol/(min mg protein), respectively. To increase the oxidation rate and alter the oxidation regiospecificity of nitro aromatics as well as to study the role of the active site residues I100, Q141, T201, and F205 of the alpha hydroxylase fragment of ToMO (TouA), DNA shuffling and saturation mutagenesis were used to generate random mutants. The mutants were initially identified by screening via a rapid agar plate assay and then were further examined by high-performance liquid chromatography (HPLC) and gas chromatography (GC). Several mutants with higher rates of activities and with different regiospecificities were identified; for example, Escherichia coli TG1 cells expressing either TouA mutant M180T/E284G or E214G/D312N/M399V produce 4-NC 4.5- and 20-fold faster than wild-type ToMO (0.037 and 0.16 nmol/min mg protein from p-NP, respectively). TouA mutant A107T/E214A had the regiospecificity of NB changed significantly from 28% to 79% p-NP. From 200 microM NB, TouA variants A101T/M114T, A110T/E392D, M180T/E284G, and E214G/D312N/M399V produce 4-NC whereas wild-type ToMO does not. From m-NP, TouA mutant I100Q produces 4-NC (37%) and NHQ (63%), whereas wild-type ToMO produces only 4-NC (100%). Variant A107T/E214A acts like a para enzyme and forms p-cresol as the major product (93%) from toluene with enhanced activity (2.3-fold), whereas wild-type ToMO forms 32%, 21%, and 47% of o-, m-, and p-cresol, respectively. Hence, the non-specific ToMO was converted into a regiospecific enzyme, which rivals toluene 4-monooxygenase of P. mendocina KR1 and toluene o-monooxygenase of Burkholderia cepacia G4 in its specificity.     

Purification of native and recombinant cobra venom factor using thiophilic adsorption chromatography

The complement activating venom component Cobra Venom Factor (CVF) forms a stable CVF-dependent C3 convertase complex, which initiates continuous activation of the complement system, consumes all downstream complement components and obliterates functional complement. Therefore, native CVF is routinely used as decomplementing agent in vivo and in vitro. However, in most countries, CVF and even unfractionated cobra venom are now becoming unavailable due to the CITES agreement. Although CVF is a complex molecule with three disulfide linked polypeptide chains and pronounced glycosylation, recombinant expression of the active molecule in eukaryotic host cells may provide an alternative source. In this study we describe a strategy for the production and efficient isolation of recombinant CVF from supernatant of mammalian cells. Thiophilic adsorption chromatography (TAC), an efficient procedure for purification of the human homologue C3, was evaluated for its suitability regarding purification of both native as well as recombinant CVF. Native CVF could be purified by TAC in a one-step procedure from cobra venom with yields of 92% compared to 35% by conventional approaches. After establishment of stably transfected mammalian cells recombinant CVF could be obtained and enriched from CHO supernatants by TAC to a purity of 73%, and up to 90% if an additional affinity chromatography step was included. Subsequent characterization revealed comparable hemolytic and bystander lysis activity and of rCVF and nCVF. These data demonstrate that the functional expression in mammalian cells in combination with TAC for purification renders rCVF a highly attractive substitute for its native counterpart.     

The Toluene o-Xylene Monooxygenase Enzymatic Activity for the Biosynthesis of Aromatic Antioxidants

Monocyclic phenols and catechols are important antioxidant compounds for the food and pharmaceutic industries; their production through biotransformation of low-added value starting compounds is of major biotechnological interest. The toluene o-xylene monooxygenase (ToMO) from Pseudomonas sp. OX1 is a bacterial multicomponent monooxygenase (BMM) that is able to hydroxylate a wide array of aromatic compounds and has already proven to be a versatile biochemical tool to produce mono- and dihydroxylated derivatives of aromatic compounds. The molecular determinants of its regioselectivity and substrate specificity have been thoroughly investigated, and a computational strategy has been developed which allows designing mutants able to hydroxylate non-natural substrates of this enzyme to obtain high-added value compounds of commercial interest. In this work, we have investigated the use of recombinant ToMO, expressed in cells of Escherichia coli strain JM109, for the biotransformation of non-natural substrates of this enzyme such as 2-phenoxyethanol, phthalan and 2-indanol to produce six hydroxylated derivatives. The hydroxylated products obtained were identified, isolated and their antioxidant potential was assessed both in vitro, using the DPPH assay, and on the rat cardiomyoblast cell line H9c2. Incubation of H9c2 cells with the hydroxylated compounds obtained from ToMO-catalyzed biotransformation induced a differential protective effect towards a mild oxidative stress induced by the presence of sodium arsenite. The results obtained confirm once again the versatility of the ToMO system for oxyfunctionalization reactions of biotechnological importance. Moreover, the hydroxylated derivatives obtained possess an interesting antioxidant potential that encourages the use of the enzyme for further functionalization reactions and their possible use as scaffolds to design novel bioactive molecules.     

Crystal structure of the toluene/o-xylene monooxygenase hydroxylase from Pseudomonas stutzeri OX1. Insight into the substrate specificity, substrate channeling, and active site tuning of multicomponent monooxygenases

The four-component toluene/o-xylene monooxygenase (ToMO) from Pseudomonas stutzeri OX1 is capable of oxidizing arenes, alkenes, and haloalkanes at a carboxylate-bridged diiron center similar to that of soluble methane monooxygenase (sMMO). The remarkable variety of substrates accommodated by ToMO invites applications ranging from bioremediation to the regio- and enantiospecific oxidation of hydrocarbons on an industrial scale. We report here the crystal structures of the ToMO hydroxylase (ToMOH), azido ToMOH, and ToMOH containing the product analogue 4-bromophenol to 2.3 A or greater resolution. The catalytic diiron(III) core resembles that of the sMMO hydroxylase, but aspects of the alpha2beta2gamma2 tertiary structure are notably different. Of particular interest is a 6-10 A-wide channel of approximately 35 A in length extending from the active site to the protein surface. The presence of three bromophenol molecules in this space confirms this route as a pathway for substrate entrance and product egress. An analysis of the ToMOH active site cavity offers insights into the different substrate specificities of multicomponent monooxygenases and explains the behavior of mutant forms of homologous enzymes described in the literature.     

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.     

Molecular Determinants of the Regioselectivity of Toluene/o-Xylene Monooxygenase from Pseudomonas sp. Strain OX1

Bacterial multicomponent monooxygenases (BMMs) are a heterogeneous family of di-iron monooxygenases which share the very interesting ability to hydroxylate aliphatic and/or aromatic hydrocarbons. Each BMM possesses defined substrate specificity and regioselectivity which match the metabolic requirements of the strain from which it has been isolated. Pseudomonas sp. strain OX1, a strain able to metabolize o-, m-, and p-cresols, produces the BMM toluene/o-xylene monooxygenase (ToMO), which converts toluene to a mixture of o-, m-, and p-cresol isomers. In order to investigate the molecular determinants of ToMO regioselectivity, we prepared and characterized 15 single-mutant and 3 double-mutant forms of the ToMO active site pocket. Using the Monte Carlo approach, we prepared models of ToMO-substrate and ToMO-reaction intermediate complexes which allowed us to provide a molecular explanation for the regioselectivities of wild-type and mutant ToMO enzymes. Furthermore, using binding energy values calculated by energy analyses of the complexes and a simple mathematical model of the hydroxylation reaction, we were able to predict quantitatively the regioselectivities of the majority of the variant proteins with good accuracy. The results show not only that the fine-tuning of ToMO regioselectivity can be achieved through a careful alteration of the shape of the active site but also that the effects of the mutations on regioselectivity can be quantitatively predicted a priori.     

Phenol Hydroxylase and Toluene/o-Xylene Monooxygenase from Pseudomonas stutzeri OX1: Interplay between Two Enzymes

Degradation of aromatic hydrocarbons by aerobic bacteria is generally divided into an upper pathway, which produces dihydroxylated aromatic intermediates by the action of monooxygenases, and a lower pathway, which processes these intermediates down to molecules that enter the citric acid cycle. Bacterial multicomponent monooxygenases (BMMs) are a family of enzymes divided into six distinct groups. Most bacterial genomes code for only one BMM, but a few cases (3 out of 31) of genomes coding for more than a single monooxygenase have been found. One such case is the genome of Pseudomonas stutzeri OX1, in which two different monooxygenases have been found, phenol hydroxylase (PH) and toluene/o-xylene monooxygenase (ToMO). We have already demonstrated that ToMO is an oligomeric protein whose subunits transfer electrons from NADH to oxygen, which is eventually incorporated into the aromatic substrate. However, no molecular data are available on the structure and on the mechanism of action of PH. To understand the metabolic significance of the association of two similar enzymatic activities in the same microorganism, we expressed and characterized this novel phenol hydroxylase. Our data indicate that the PH P component of PH transfers electrons from NADH to a subcomplex endowed with hydroxylase activity. Moreover, a regulatory function can be suggested for subunit PH M. Data on the specificity and the kinetic constants of ToMO and PH strongly support the hypothesis that coupling between the two enzymatic systems optimizes the use of nonhydroxylated aromatic molecules by the draining effect of PH on the product(s) of oxidation catalyzed by ToMO, thus avoiding phenol accumulation.     

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.     

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.     

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%).     

Phenol hydroxylase and toluene/o-xylene monooxygenase from Pseudomonas stutzeri OX1: interplay between two enzymes

Degradation of aromatic hydrocarbons by aerobic bacteria is generally divided into an upper pathway, which produces dihydroxylated aromatic intermediates by the action of monooxygenases, and a lower pathway, which processes these intermediates down to molecules that enter the citric acid cycle. Bacterial multicomponent monooxygenases (BMMs) are a family of enzymes divided into six distinct groups. Most bacterial genomes code for only one BMM, but a few cases (3 out of 31) of genomes coding for more than a single monooxygenase have been found. One such case is the genome of Pseudomonas stutzeri OX1, in which two different monooxygenases have been found, phenol hydroxylase (PH) and toluene/o-xylene monooxygenase (ToMO). We have already demonstrated that ToMO is an oligomeric protein whose subunits transfer electrons from NADH to oxygen, which is eventually incorporated into the aromatic substrate. However, no molecular data are available on the structure and on the mechanism of action of PH. To understand the metabolic significance of the association of two similar enzymatic activities in the same microorganism, we expressed and characterized this novel phenol hydroxylase. Our data indicate that the PH P component of PH transfers electrons from NADH to a subcomplex endowed with hydroxylase activity. Moreover, a regulatory function can be suggested for subunit PH M. Data on the specificity and the kinetic constants of ToMO and PH strongly support the hypothesis that coupling between the two enzymatic systems optimizes the use of nonhydroxylated aromatic molecules by the draining effect of PH on the product(s) of oxidation catalyzed by ToMO, thus avoiding phenol accumulation.