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  • Wood, Thomas K.  (9)
  • 1
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    Saturation Mutagenesis of Toluene ortho -Monooxygenase of Burkholderia cepacia G4 for Enhanced 1-Naphthol Synthesis and Chloroform Degradation
    American Society for Microbiology ; 2004
    In:  Applied and Environmental Microbiology Vol. 70, No. 6 ( 2004-06), p. 3246-3252
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 70, No. 6 ( 2004-06), p. 3246-3252
    Abstract: Directed evolution of toluene ortho -monooxygenase (TOM) of Burkholderia cepacia G4 previously created the hydroxylase α-subunit (TomA3) V106A variant (TOM-Green) with increased activity for both trichloroethylene degradation (twofold enhancement) and naphthalene oxidation (six-times-higher activity). In the present study, saturation mutagenesis was performed at position A106 with Escherichia coli TG1/pBS(Kan)TOMV106A to improve TOM activity for both chloroform degradation and naphthalene oxidation. Whole cells expressing the A106E variant had two times better naphthalene-to-1-naphthol activity than the wild-type cells ( V max of 9.3 versus 4.5 nmol · min −1  · mg of protein −1 and unchanged K m ), and the regiospecificity of the A106E variant was unchanged, with 98% 1-naphthol formed, as was confirmed with high-pressure liquid chromatography. The A106E variant degrades its natural substrate toluene 63% faster than wild-type TOM does (2.12 ± 0.07 versus 1.30 ± 0.06 nmol · min −1  · mg of protein −1 [mean ± standard deviation]) at 91 μM and has a substantial decrease in regiospecificity, since o -cresol (50%), m -cresol (25%), and p -cresol (25%) are formed, in contrast to the 98% o -cresol formed by wild-type TOM. The A106E variant also has an elevated expression level compared to that of wild-type TOM, as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Another variant, the A106F variant, has 2.8-times-better chloroform degradation activity based on gas chromatography ( V max of 2.61 versus 0.95 nmol · min −1  · mg of protein −1 and unchanged K m ) and chloride release (0.034 ± 0.002 versus 0.012 ± 0.001 nmol · min −1  · mg of protein −1 ). The A106F variant also was expressed at levels similar to those of wild-type TOM and 62%-better toluene oxidation activity than wild-type TOM (2.11 ± 0.3 versus 1.30 ± 0.06 nmol · min −1  · mg of protein −1 ). A shift in regiospecificity of toluene hydroxylation was also observed for the A106F variant, with o -cresol (28%), m -cresol (18%), and p -cresol (54%) being formed. Statistical analysis was used to estimate that 292 colonies must be screened for a 99% probability that all 64 codons were sampled during saturation mutagenesis.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.70.6.3246-3252.2004
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2004
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  • 2
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    Toluene 3-Monooxygenase of Ralstonia pickettii PKO1 Is a para -Hydroxylating Enzyme
    American Society for Microbiology ; 2004
    In:  Journal of Bacteriology Vol. 186, No. 10 ( 2004-05-15), p. 3117-3123
    Details
    In: Journal of Bacteriology, American Society for Microbiology, Vol. 186, No. 10 ( 2004-05-15), p. 3117-3123
    Abstract: 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 K m value of 250 μM 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. 1 H nuclear magnetic resonance analysis confirmed the structural identity of p -cresol in samples containing the product of hydroxylation of toluene by pBS(Kan)T3MO.
    Type of Medium: Online Resource
    ISSN: 0021-9193 , 1098-5530
    URL: Article
    DOI: 10.1128/JB.186.10.3117-3123.2004
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2004
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  • 3
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    Protein Engineering of Toluene Monooxygenases for Synthesis of Chiral Sulfoxides
    American Society for Microbiology ; 2008
    In:  Applied and Environmental Microbiology Vol. 74, No. 5 ( 2008-03), p. 1555-1566
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 74, No. 5 ( 2008-03), p. 1555-1566
    Abstract: 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.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.01849-07
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2008
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    detail.hit.zdb_id: 1478346-0
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  • 4
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    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
    American Society for Microbiology ; 2004
    In:  Applied and Environmental Microbiology Vol. 70, No. 7 ( 2004-07), p. 3814-3820
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 70, No. 7 ( 2004-07), p. 3814-3820
    Abstract: 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).
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.70.7.3814-3820.2004
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2004
    detail.hit.zdb_id: 223011-2
    detail.hit.zdb_id: 1478346-0
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  • 5
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    Physiological relevance of successive hydroxylations of toluene by toluene para -monooxygenase of Ralstonia pickettii PKO1
    Informa UK Limited ; 2004
    In:  Biocatalysis and Biotransformation Vol. 22, No. 4 ( 2004-07), p. 283-289
    Details
    In: Biocatalysis and Biotransformation, Informa UK Limited, Vol. 22, No. 4 ( 2004-07), p. 283-289
    Type of Medium: Online Resource
    ISSN: 1024-2422 , 1029-2446
    URL: Article
    DOI: 10.1080/10242420400012008
    Language: English
    Publisher: Informa UK Limited
    Publication Date: 2004
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  • 6
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    Protein engineering of toluene 4‐monooxygenase of Pseudomonas mendocina KR1 for synthesizing 4‐nitrocatechol from nitrobenzene
    Wiley ; 2004
    In:  Biotechnology and Bioengineering Vol. 87, No. 6 ( 2004-09-20), p. 779-790
    Details
    In: Biotechnology and Bioengineering, Wiley, Vol. 87, No. 6 ( 2004-09-20), p. 779-790
    Abstract: After discovering that toluene 4‐monooxygenase (T4MO) of Pseudomonas mendocina KR1 oxidizes nitrobenzene to 4‐nitrocatechol, albeit at a very low rate, this reaction was improved using directed evolution and saturation mutagenesis. Screening 550 colonies from a random mutagenesis library generated by error‐prone PCR of tmoAB using Escherichia coli TG1/pBS(Kan)T4MO on agar plates containing nitrobenzene led to the discovery of nitrocatechol‐producing mutants. One mutant, NB1, contained six amino acid substitutions (TmoA Y22N, I84Y, S95T, I100S, S400C; TmoB D79N). It was believed that position I100 of the α subunit of the hydroxylase (TmoA) is the most significant for the change in substrate reactivity due to previous results in our lab with a similar enzyme, toluene ortho ‐monooxygenase of Burkholderia cepacia G4. Saturation mutagenesis at this position resulted in the generation of two more nitrocatechol mutants, I100A and I100S; the rate of 4‐nitrocatechol formation by I100A was more than 16 times higher than that of wild‐type T4MO at 200 μ M nitrobenzene (0.13 ± 0.01 vs. 0.008 ± 0.001 nmol/min·mg protein). HPLC and mass spectrometry analysis revealed that variants NB1, I100A, and I100S produce 4‐nitrocatechol via m ‐nitrophenol, while the wild‐type produces primarily p ‐nitrophenol and negligible amounts of nitrocatechol. Relative to wild‐type T4MO, whole cells expressing variant I100A convert nitrobenzene into m ‐nitrophenol with a V max of 0.61 ± 0.037 vs. 0.16 ± 0.071 nmol/min·mg protein and convert m ‐nitrophenol into nitrocatechol with a V max of 3.93 ± 0.26 vs. 0.58 ± 0.033 nmol/min·mg protein. Hence, the regiospecificity of nitrobenzene oxidation was changed by the random mutagenesis, and this led to a significant increase in 4‐nitrocatechol production. The regiospecificity of toluene oxidation was also altered, and all of the mutants produced 20% m ‐cresol and 80% p ‐cresol, while the wild‐type produces 96% p ‐cresol. Interestingly, the rate of toluene oxidation (the natural substrate of the enzyme) by I100A was also higher by 65% (7.2 ± 1.2 vs. 4.4 ± 0.3 nmol/min mg protein). Homology‐based modeling of TmoA suggests reducing the size of the side chain of I100 leads to an increase in the width of the active site channel, which facilitates access of substrates and promotes more flexible orientations. © 2004 Wiley Periodicals, Inc.
    Type of Medium: Online Resource
    ISSN: 0006-3592 , 1097-0290
    URL: Issue
    URL: Article
    DOI: 10.1002/bit.v87:6
    DOI: 10.1002/bit.20185
    Language: English
    Publisher: Wiley
    Publication Date: 2004
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  • 7
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    Controlling the Regiospecific Oxidation of Aromatics via Active Site Engineering of Toluene para-Monooxygenase of Ralstonia pickettii PKO1
    Elsevier BV ; 2005
    In:  Journal of Biological Chemistry Vol. 280, No. 1 ( 2005-01), p. 506-514
    Details
    In: Journal of Biological Chemistry, Elsevier BV, Vol. 280, No. 1 ( 2005-01), p. 506-514
    Type of Medium: Online Resource
    ISSN: 0021-9258
    URL: Article
    DOI: 10.1074/jbc.M410320200
    Language: English
    Publisher: Elsevier BV
    Publication Date: 2005
    detail.hit.zdb_id: 2141744-1
    detail.hit.zdb_id: 1474604-9
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  • 8
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    Rapid Methods for High-Throughput Detection of Sulfoxides
    American Society for Microbiology ; 2009
    In:  Applied and Environmental Microbiology Vol. 75, No. 14 ( 2009-07-15), p. 4711-4719
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 75, No. 14 ( 2009-07-15), p. 4711-4719
    Abstract: Enantiopure sulfoxides are prevalent in drugs and are useful chiral auxiliaries in organic synthesis. The biocatalytic enantioselective oxidation of prochiral sulfides is a direct and economical approach for the synthesis of optically pure sulfoxides. The selection of suitable biocatalysts requires rapid and reliable high-throughput screening methods. Here we present four different methods for detecting sulfoxides produced via whole-cell biocatalysis, three of which were exploited for high-throughput screening. Fluorescence detection based on the acid activation of omeprazole was utilized for high-throughput screening of mutant libraries of toluene monooxygenases, but no active variants have been discovered yet. The second method is based on the reduction of sulfoxides to sulfides, with the coupled release and measurement of iodine. The availability of solvent-resistant microtiter plates enabled us to modify the method to a high-throughput format. The third method, selective inhibition of horse liver alcohol dehydrogenase, was used to rapidly screen highly active and/or enantioselective variants at position V106 of toluene ortho -monooxygenase in a saturation mutagenesis library, using methyl- p -tolyl sulfide as the substrate. A success rate of 89% (i.e., 11% false positives) was obtained, and two new mutants were selected. The fourth method is based on the colorimetric detection of adrenochrome, a back-titration procedure which measures the concentration of the periodate-sensitive sulfide. Due to low sensitivity during whole-cell screening, this method was found to be useful only for determining the presence or absence of sulfoxide in the reaction. The methods described in the present work are simple and inexpensive and do not require special equipment.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.02674-08
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2009
    detail.hit.zdb_id: 223011-2
    detail.hit.zdb_id: 1478346-0
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  • 9
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    Altering Toluene 4-Monooxygenase by Active-Site Engineering for the Synthesis of 3-Methoxycatechol, Methoxyhydroquinone, and Methylhydroquinone
    American Society for Microbiology ; 2004
    In:  Journal of Bacteriology Vol. 186, No. 14 ( 2004-07-15), p. 4705-4713
    Details
    In: Journal of Bacteriology, American Society for Microbiology, Vol. 186, No. 14 ( 2004-07-15), p. 4705-4713
    Abstract: Wild-type toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1 oxidizes toluene to p -cresol (96%) and oxidizes benzene sequentially to phenol, to catechol, and to 1,2,3-trihydroxybenzene. In this study T4MO was found to oxidize o -cresol to 3-methylcatechol (91%) and methylhydroquinone (9%), to oxidize m -cresol and p -cresol to 4-methylcatechol (100%), and to oxidize o -methoxyphenol to 4-methoxyresorcinol (87%), 3-methoxycatechol (11%), and methoxyhydroquinone (2%). Apparent V max values of 6.6 ± 0.9 to 10.7 ± 0.1 nmol/min/ mg of protein were obtained for o -, m -, and p -cresol oxidation by wild-type T4MO, which are comparable to the toluene oxidation rate (15.1 ± 0.8 nmol/min/mg of protein). After these new reactions were discovered, saturation mutagenesis was performed near the diiron catalytic center at positions I100, G103, and A107 of the alpha subunit of the hydroxylase (TmoA) based on directed evolution of the related toluene o- monooxygenase of Burkholderia cepacia G4 (K. A. Canada, S. Iwashita, H. Shim, and T. K. Wood, J. Bacteriol. 184 : 344-349, 2002) and a previously reported T4MO G103L regiospecific mutant (K. H. Mitchell, J. M. Studts, and B. G. Fox, Biochemistry 41 : 3176-3188, 2002). By using o -cresol and o -methoxyphenol as model substrates, regiospecific mutants of T4MO were created; for example, TmoA variant G103A/A107S produced 3-methylcatechol (98%) from o -cresol twofold faster and produced 3-methoxycatechol (82%) from 1 mM o -methoxyphenol seven times faster than the wild-type T4MO (1.5 ± 0.2 versus 0.21 ± 0.01 nmol/min/mg of protein). Variant I100L produced 3-methoxycatechol from o -methoxyphenol four times faster than wild-type T4MO, and G103S/A107T produced methylhydroquinone (92%) from o -cresol fourfold faster than wild-type T4MO and there was 10 times more in terms of the percentage of the product. Variant G103S produced 40-fold more methoxyhydroquinone from o -methoxyphenol than the wild-type enzyme produced (80 versus 2%) and produced methylhydroquinone (80%) from o -cresol. Hence, the regiospecific oxidation of o -methoxyphenol and o -cresol was changed for significant synthesis of 3-methoxycatechol, methoxyhydroquinone, 3-methylcatechol, and methylhydroquinone. The enzyme variants also demonstrated altered monohydroxylation regiospecificity for toluene; for example, G103S/A107G formed 82% o -cresol, so saturation mutagenesis converted T4MO into an ortho -hydroxylating enzyme. Furthermore, G103S/A107T formed 100% p -cresol from toluene; hence, a better para- hydroxylating enzyme than wild-type T4MO was formed. Structure homology modeling suggested that hydrogen bonding interactions of the hydroxyl groups of altered residues S103, S107, and T107 influence the regiospecificity of the oxygenase reaction.
    Type of Medium: Online Resource
    ISSN: 0021-9193 , 1098-5530
    URL: Article
    DOI: 10.1128/JB.186.14.4705-4713.2004
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2004
    detail.hit.zdb_id: 1481988-0
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