Expression and purification of the recombinant subunits of toluene/<i>o</i>‐xylene monooxygenase and reconstitution of the active complex

Cafaro, Valeria; Scognamiglio, Roberta; Viggiani, Ambra; Izzo, Viviana; Passaro, Irene; Notomista, Eugenio; Piaz, Fabrizio Dal; Amoresano, Angela; Casbarra, Annarita; Pucci, Piero; Donato, Alberto Di

doi:10.1046/j.1432-1033.2002.03281.x

Cited by 69 publications

(108 citation statements)

References 43 publications

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“…Complete reduction yielded a spectrum (dashed line) with a shoulder centered at ∼ 520 nm consistent with other known NADH oxidoreductases. The purified protein had A 280 /A 460 of ∼3.5, which is comparable to that observed in the purified oxidoreductases from toluene o-xylene monooxygenase, pthalate dioxygenase reductase and phenol hydroxylase [20,21]. The spectrum of the oxidized form of the enzyme shown in Fig.…”

Section: Characterization Of T4mofsupporting

confidence: 67%

“…Quantification of FAD content revealed that the purified T4moF contained 0.9 ± 0.1 mol of FAD per mol of protein. TomoF and MMOR, the NADH oxidoreductases from the related diiron enzymes toluene/o-xylene monooxygenase and methane monooxygenase, respectively, also contain FAD [20,22].…”

Section: Characterization Of T4mofmentioning

confidence: 99%

“…Recently, a purification of TomoF, the oxidoreductase of the closely related toluene/o-xylene hydroxylase was reported [20]. This purification included solubilization of the protein from inclusion bodies, refolding, and then reconstitution of the iron sulfur cluster and flavin cofactors.…”

Section: Purificationmentioning

confidence: 99%

“…This purification included solubilization of the protein from inclusion bodies, refolding, and then reconstitution of the iron sulfur cluster and flavin cofactors. This successful, but labor-intensive effort, gave ∼3-4 mg of protein per 10 mg of inclusion bodies prepared [20].…”

Section: Purificationmentioning

confidence: 99%

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Soluble expression and purification of the oxidoreductase component of toluene 4-monooxygenase

Bailey

Elsen

Pierce

et al. 2008

Protein Expression and Purification

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Toluene 4-monooxygenase (T4MO) is a member of the bacterial multicomponent monooxygenases, an enzyme family that utilizes a soluble diiron hydroxylase to oxidize a variety of hydrocarbons as the initial step in their metabolism. The hydroxylases obtain reducing equivalents from NAD(P)H via an electron transfer chain that is initiated by an oxidoreductase containing an N-terminal ferredoxin domain and C-terminal flavin-and NAD-binding domains. T4moF, the NADH oxidoreductase of T4MO, was expressed as a soluble protein in Escherichia coli BL21(DE3) from the pUC-derived expression vector pRS205. This vector contains a lac promoter instead of a T7 promoter. A three step purification from the soluble cell lysate yielded ∼1 mg of T4moF per gram of wet cell paste with greater than 90% purity. The purified protein contained 1 mol of FAD and 2 mol of Fe per mol of T4moF; quantitiative EPR spectroscopy showed ∼1 mol of the S = ½ signal from the reduced [2Fe-2S] cluster per mol of T4moF. Steady state kinetic analysis of p-cresol formation activity treating T4moF as the variable substrate while all other proteins and substrates were held constant gave apparent K M -and apparent k cat -values of 0.15 μM and 3.0 s -1 , respectively. This expression system and purification allows for the recovery of the soluble oxidoreductase in yields that facilitate further biochemical and structural characterizations.

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Section: Characterization Of T4mofsupporting

confidence: 67%

Section: Characterization Of T4mofmentioning

confidence: 99%

Section: Purificationmentioning

confidence: 99%

Section: Purificationmentioning

confidence: 99%

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Soluble expression and purification of the oxidoreductase component of toluene 4-monooxygenase

Bailey

Elsen

Pierce

et al. 2008

Protein Expression and Purification

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“…Expression and Purification of ToMOH-ToMOH was expressed from the plasmid pET22BEA in BL21(DE3) E. coli cells (17). Cells were grown at 37°C in 10 2-liter baffled flasks containing 1 liter of LB media.…”

Section: Methodsmentioning

confidence: 99%

Crystal Structure of the Toluene/o-Xylene Monooxygenase Hydroxylase from Pseudomonas stutzeri OX1

Sazinsky

Bard²,

Donato

et al. 2004

Journal of Biological Chemistry

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188

177

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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 regioand 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 Å or greater resolution. The catalytic diiron(III) core resembles that of the sMMO hydroxylase, but aspects of the ␣ 2 ␤ 2 ␥ 2 tertiary structure are notably different. Of particular interest is a 6 -10 Å-wide channel of ϳ35 Å 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.Bacterial multicomponent monooxygenases (BMMs) 1 comprise a family of carboxylate-bridged non-heme diiron enzymes capable of oxidizing a broad range of hydrocarbons including C 1 -C 8 alkanes, alkenes, and aromatics (1, 2). Four characterized subclasses of multicomponent monooxygenases have been defined (2, 3). These are soluble methane monooxygenases (sMMOs), four-component alkene/arene monooxygenases or toluene monooxygenases (TMOs), three-component phenol hydroxylases (PHs), and ␣␤ alkene monoxygenases (AMOs), of which all are believed to have evolved from a common ancestor. Bacteria containing multicomponent monooxygenases are capable of using specific hydrocarbon substrates as their primary source of carbon and energy (1, 2, 4). The remarkable range of substrate specificity exhibited by these enzymes endows these bacteria with the ability to bioremediate environmentally harmful substances such as trichloroethylene and petroleum spills (5, 6) and to regulate the global carbon cycle (4). BMMs can also perform regio-and stereospecific hydroxylations, making them useful for producing pure feedstocks for industrial synthesis (7). These enzyme systems, although highly homologous, have evolved different substrate specificities. Only soluble methane monooxygenase can activate the inert C-H bond of methane, which is one of the most difficult reactions to perform in nature (1), whereas the catalytic abilities of TMOs are limited to aromatics, alkenes, and some haloalkanes (2, 5).Substrate hydroxylation in BMMs occurs at a dioxygen-activated, carboxylate-bridged diiron center in the ␣-subunit of a ϳ220 -250 kDa hydroxylase component that is an (␣␤␥) 2 heterodimer or, in the case of one known AMO, an ␣␤ monomer (1-3, 8, 9). Sequence identity comparisons and spectroscopic studies suggest that the diiron centers of...

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

Tao

Bentley

Wood

2005

Biotech & Bioengineering

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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.

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Expression and purification of the recombinant subunits of toluene/o‐xylene monooxygenase and reconstitution of the active complex

Cited by 69 publications

References 43 publications

Soluble expression and purification of the oxidoreductase component of toluene 4-monooxygenase

Soluble expression and purification of the oxidoreductase component of toluene 4-monooxygenase

Crystal Structure of the Toluene/o-Xylene Monooxygenase Hydroxylase from Pseudomonas stutzeri OX1

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

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