TOL plasmid-specified xylene oxygenase is a wide substrate range monooxygenase capable of olefin epoxidation

Wubbolts, Marcel; Reuvekamp, Peter; Witholt, Bernard

doi:10.1016/0141-0229(94)90127-9

Cited by 65 publications

(48 citation statements)

References 32 publications

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“…These respective alcohols are further metabolized to benzoate and m-and p-toluates via their corresponding aldehydes before being subjected to meta cleavage. On the other hand, o-xylene has not been accepted as a substrate for XylMA (40), although the reason for this has not been elucidated.…”

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confidence: 98%

Isolation and Characterization of o -Xylene Oxygenase Genes from Rhodococcus opacus TKN14

Maruyama

Ishikura

Taki

et al. 2005

Appl Environ Microbiol

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o-Xylene is one of the most difficult-to-degrade environmental pollutants. We report here Rhodococcus genes mediating oxygenation in the first step of o-xylene degradation. Rhodococcus opacus TKN14, isolated from soil contaminated with o-xylene, was able to utilize o-xylene as the sole carbon source and to metabolize it to o-methylbenzoic acid. A cosmid library from the genome of this strain was constructed in Escherichia coli. A bioconversion analysis revealed that a cosmid clone incorporating a 15-kb NotI fragment had the ability to convert o-xylene into o-methylbenzyl alcohol. The sequence analysis of this 15-kb region indicated the presence of a gene cluster significantly homologous to the naphthalene-inducible dioxygenase gene clusters (nidABCD) that had been isolated from Rhodococcus sp. strain I24. Complementation studies, using E. coli expressing various combinations of individual open reading frames, revealed that a gene (named nidE) for rubredoxin (Rd) and a novel gene (named nidF) encoding an auxiliary protein, which had no overall homology with any other proteins, were indispensable for the methyl oxidation reaction of o-xylene, in addition to the dioxygenase iron-sulfur protein genes (nidAB). Regardless of the presence of NidF, the enzyme composed of NidABE was found to function as a typical naphthalene dioxygenase for converting naphthalene and various (di)methylnaphthalenes into their corresponding cis-dihydrodiols. All the nidABEF genes were transcriptionally induced in R. opacus TKN14 by the addition of o-xylene to a mineral salt medium. It is very likely that these genes are involved in the degradation pathways of a wide range of aromatic hydrocarbons by Rhodococcus species as the first key enzyme. o-, m-and p-xylenes, which are included in petroleum, are used as solvents in the production of chemicals and drugs, as thinners for paints and enamels, and as intermediates for the synthesis of numerous chemical compounds. They are therefore widespread environmental contaminants in groundwater and soil (4,11,27). In m-and p-xylenes, numerous bacteria that are able to utilize them as the sole carbon and energy source have been isolated, and details of their catabolic pathways have been well documented. The most elucidated examples are the catabolism of toluene, m-xylene, and p-xylene with the enzymes encoded on a transmissible TOL plasmid (pWW0) from Pseudomonas putida mt-2 (38, 39). Xylene monooxygenase is the first enzyme on this degradation pathway (14, 34). This enzyme consists of two polypeptide subunits encoded by the xylM and xylA genes. XylA, the NADH acceptor reductase component (34), has been characterized as an electron transport protein transferring reducing equivalents from NADH to XylM, which is the hydroxylase component located in the membrane (18). Toluene and m-and p-xylenes are, respectively, metabolized with XylMA to benzyl alcohol and m-and p-methylbenzyl alcohol. These respective alcohols are further metabolized to benzoate and m-and p-toluates via their corresponding aldehydes b...

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Isolation and Characterization of o -Xylene Oxygenase Genes from Rhodococcus opacus TKN14

Maruyama

Ishikura

Taki

et al. 2005

Appl Environ Microbiol

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“…XMO expressed in Escherichia coli oxidizes toluene and xylenes but also m-and p-ethyl-, methoxy-, nitro-, and chloro-substituted toluenes, as well as m-bromo-substituted toluene, to the corresponding benzyl alcohol derivatives (21,65). Furthermore, styrene is transformed into S-styrene oxide with an enantiomeric excess (ee) of 95% (64,65). The one-step oxygenation of styrene catalyzed by recombinant XMO in growing cells of E. coli was applied to produce S-styrene oxide on a 2-liter scale with hexadecane as the second organic phase (30).…”

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Characterization and Application of Xylene Monooxygenase for Multistep Biocatalysis

Bühler

Witholt

Hauer

et al. 2002

Appl Environ Microbiol

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Xylene monooxygenase of Pseudomonas putida mt-2 catalyzes multistep oxidations of one methyl group of toluene and xylenes. Recombinant Escherichia coli expressing the monooxygenase genes xylM and xylA catalyzes the oxygenation of toluene, pseudocumene, the corresponding alcohols, and the corresponding aldehydes, all by a monooxygenation type of reaction (B. Bühler, A. Schmid, B. Hauer, and B. Witholt, J. Biol. Chem. 275: 10085-10092, 2000). Using E. coli expressing xylMA, we investigated the kinetics of this one-enzyme three-step biotransformation. We found that unoxidized substrates like toluene and pseudocumene inhibit the second and third oxygenation steps and that the corresponding alcohols inhibit the third oxygenation step. These inhibitions might promote the energetically more favorable alcohol and aldehyde dehydrogenations in the wild type. Growth of E. coli was strongly affected by low concentrations of pseudocumene and its products. Toxicity and solubility problems were overcome by the use of a two-liquid-phase system with bis(2-ethylhexyl)phthalate as the carrier solvent, allowing high overall substrate and product concentrations. In a fed-batch-based twoliquid-phase process with pseudocumene as the substrate, we observed the consecutive accumulation of aldehyde, acid, and alcohol. Our results indicate that, depending on the reaction conditions, product formation could be directed to one specific product.During the last few decades, the microbial degradation pathways of aromatic and aliphatic hydrocarbons have received a lot of scientific interest because of the high potential of the enzyme systems involved for environmental (43) and preparative applications (38, 59). These pathways are usually initiated by an oxygenase-catalyzed chemo-, regio-, and stereoselective hydroxylation of the hydrocarbons, a reaction for which often no organic chemical counterpart is known (9, 13).The xylene degradation pathway of Pseudomonas putida mt-2 and its initiating oxygenase, the xylene monooxygenase (XMO), are among the best-studied examples of aromatic hydrocarbon degradation (5,36,57,61). The enzymes for xylene degradation are encoded on a catabolic plasmid, the TOL plasmid pWW0. XMO is the first enzyme in the upper degradation pathway for toluene and xylenes, in which a carboxylic acid is formed (1,16,58). The upper pathway also involves benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase, which catalyze the oxidation of benzyl alcohols via benzaldehydes to benzoic acids (46-48). The carboxylic acid is then transformed to substrates of the Krebs cycle through the meta cleavage pathway (10,14,36,55).XMO consists of two polypeptide subunits, encoded by xylM and xylA (16,52). XylA, the NADH:acceptor reductase component, is an electron transport protein transferring reducing equivalents from NADH to XylM (45). XylM, the hydroxylase component, is located in the membrane, and its activity depends on phospholipids and ferrous ion, with a pH optimum of 7 (44, 62).The substrate spectrum of XMO was investigated, wi...

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“…Indol conversion to indigo is considered representative of dioAl-Thani et al 2679 xygenase activity. The indol conversion was previously shown by aromatic ring dioxygenase from monocyclic aromatic hydrocarbon degraders (Wubbolts et al, 1994).…”

Section: Discussionmentioning

confidence: 99%

Isolation, biochemical and molecular characterization of 2-chlorophenol-degrading Bacillus isolates

Al‐Thani¹,

Abd‐El‐Haleem²,

Al-Shammri³

2007

Afr. J. Biotechnol.

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Pure cultures of 2-chlorophenol degrading bacteria were isolated from a natural enrichment that may be adapted to chlorophenols in the industrial zone at Umm-Saied city (Qatar). The bacteria were identified by 16S rDNA analysis, using PCR with universal primers. Comparative analysis of the 16S rDNA sequence (~ 550 bp) in the GenBank database revealed that these bacteria are related to the genus Bacillus. Molecular heterogeneity among 2-chlorophenol-degrading bacteria was investigated using REP-PCR chromosomal fingerprinting and correlated with antibiotic profile analysis. REP-PCR results strongly confirmed that the bacterial isolates from different Qatari soils produced different fingerprinting patterns. The distribution of phenol hydroxylase catabolic gene among examined isolates revealed that three isolates out of six yielded positive PCR products. Degradation of 2-chlorophenol was studied using theses cultures in liquid medium under aerobic conditions, at initial concentrations of 0.25 -2.5 mM 2-chlorophenol. Undegraded 2-chlorophenol was quantified by high-performance liquid chromatography (HPLC). Degradation rates by isolates could be determined at concentrations up to 1.5 mM. However, higher concentrations of 2-chlorophenol (2.5 mM) were inhibitory to cell growth.

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TOL plasmid-specified xylene oxygenase is a wide substrate range monooxygenase capable of olefin epoxidation

Cited by 65 publications

References 32 publications

Isolation and Characterization of o -Xylene Oxygenase Genes from Rhodococcus opacus TKN14

Isolation and Characterization of o -Xylene Oxygenase Genes from Rhodococcus opacus TKN14

Characterization and Application of Xylene Monooxygenase for Multistep Biocatalysis

Isolation, biochemical and molecular characterization of 2-chlorophenol-degrading Bacillus isolates

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