A Versatile High Throughput Screen for Dioxygenase Activity Using Solid-Phase Digital Imaging

Joern, John M.; Sakamoto, Takeshi; Arisawa, Akira; Arnold, Frances H.

doi:10.1177/108705710100600403

Cited by 21 publications

(15 citation statements)

References 19 publications

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“…The Gibbs reaction has been used for detection of dioxygenase activity (27), but the reported protocol was not sensitive enough to differentiate the small changes in product levels expected during directed evolution. With acid treatment prior to the Gibbs reaction (to catalyze cis-dihydrodiol dehydration to the corresponding phenol) and other modifications, we established a liquid assay system ϳ20 times more sensitive than that reported previously (19). A solid-phase version of the assay was also described (19).…”

Section: Resultsmentioning

confidence: 99%

“…With acid treatment prior to the Gibbs reaction (to catalyze cis-dihydrodiol dehydration to the corresponding phenol) and other modifications, we established a liquid assay system ϳ20 times more sensitive than that reported previously (19). A solid-phase version of the assay was also described (19). This assay is suitable for monitoring production of 3-hydroxy-4-picoline.…”

Section: Resultsmentioning

confidence: 99%

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Laboratory Evolution of Toluene Dioxygenase To Accept 4-Picoline as a Substrate

Sakamoto

Joern

Arisawa

et al. 2001

Appl Environ Microbiol

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We are using directed evolution to extend the range of dioxygenase-catalyzed biotransformations to include substrates that are either poorly accepted or not accepted at all by the naturally occurring enzymes. Here we report on the oxidation of a heterocyclic substrate, 4-picoline, by toluene dioxygenase (TDO) and improvement of the enzyme's activity by laboratory evolution. The biotransformation of 4-picoline proceeds at only ϳ4.5% of the rate of the natural reaction on toluene. Random mutagenesis, saturation mutagenesis, and screening directly for product formation using a modified Gibbs assay generated mutant TDO 3-B38, in which the wild-type stop codon was replaced with a codon encoding threonine. Escherichia coli-expressed TDO 3-B38 exhibited 5.6 times higher activity toward 4-picoline and ϳ20% more activity towards toluene than wild-type TDO. The product of the biotransformation of 4-picoline is 3-hydroxy-4-picoline; no cis-diols of 4-picoline were observed.Dioxygenase enzymes involved in the catabolism of aromatic hydrocarbons by soil microorganisms nicely illustrate nature's ability to adapt to different carbon sources through evolution of the substrate specificity of the biodegradation enzymes (35). The biodegradation pathway often begins with the dioxygenasecatalyzed regio-and enantio-specific introduction of molecular oxygen into aromatic compounds to form the corresponding arene cis-diols, with consumption of NADH (13, 36) ( Fig. 1).It has been noted that the chiral products of the dioxygenase reaction can be converted in a variety of synthetic reactions to advanced intermediates for natural-product synthesis (8, 15). Toluene dioxygenase (TDO) from Pseudomonas putida readily dihydroxylates aromatic carbocycles with one or two small hydrophobic substituents (6, 33). Activity towards much larger, fused-ring substrates or substrates with polar or bulky substituents is considerably reduced or nonexistent (33).We are attempting to extend the utility of dioxygenase-catalyzed biotransformations by engineering the catalysts to accept a wider range of substrates, including small heterocyclic compounds and polar-substituted carbocyclic aromatics. N-Heterocyclic compounds are useful for the synthesis of biologically active compounds; recent reports describe the regioand/or stereo-controlled biooxidation of N-heterocycles (20,26). Bicyclic compounds such as quinolines and benzofurans are accepted by TDO, with oxygen insertion occurring primarily in the carbocylic rings (3-5).To date, reactions on single-ring heterocycles have not been reported. We have found that TDO catalyzes the oxygenation of 4-picoline (4-methylpyridine), although at a much slower rate than on its preferred substrate toluene.A strategy of DNA shuffling to create libraries of hybrid genes and screening has been used with some success to extend the substrate range of dioxygenases that degrade environmental pollutants such as polychlorinated biphenyls (7, 21). An alternative approach is to fine tune a single gene by accumulating beneficial mutat...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Laboratory Evolution of Toluene Dioxygenase To Accept 4-Picoline as a Substrate

Sakamoto

Joern

Arisawa

et al. 2001

Appl Environ Microbiol

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“…Saturation mutagenesis was performed at positions 277 and 312 using an overlap extension PCR-based method (3). Mutants exhibiting lower activity for pABA and higher activity for Aprn were identified using Gibbs reagent-based activity assays (7,21) and were confirmed by determining whole-cell activity with Aprn. The positive mutants that exhibited higher activity for Aprn were found to be the F312A, F312S, F312V, F312L, F312I, L277A, and L277V mutants.…”

Section: Homology Modelingmentioning

confidence: 96%

Probing the Substrate Specificity of Aminopyrrolnitrin Oxygenase (PrnD) by Mutational Analysis

Lee

Ang²,

Zhao³

2006

J Bacteriol

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Molecular modeling and mutational analysis (site-directed mutagenesis and saturation mutagenesis) were used to probe the molecular determinants of the substrate specificity of aminopyrrolnitrin oxygenase (PrnD) from Pseudomonas fluorescens Pf-5. There are 17 putative substrate-contacting residues, and mutations at two of the positions, positions 312 and 277, could modulate the enzyme substrate specificity separately or in combination. Interestingly, several of the mutants obtained exhibited higher catalytic efficiency (approximately two-to sevenfold higher) with the physiological substrate aminopyrrolnitrin than the wild-type enzyme exhibited.

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“…[6,7] Furthermore, ROs were shown to be suitable for bioremediation applications and to have the ability to process xenobiotics. [8,9] In industry, ROs are utilized to manufacture the blue dye indigo, which is crucial for the production of denim cloth.…”

mentioning

confidence: 99%

“…[5] Over 300 diverse substrates ranging from polyaromatic hydrocarbons such as phenanthracene to halogenated ethylenes and flavonoid structures were reportedt ob eh ydroxylated with these enzymes. [6,7] Furthermore, ROs were shown to be suitable for bioremediation applications and to have the ability to process xenobiotics. [8,9] In industry, ROs are utilized to manufacture the blue dye indigo, which is crucial for the production of denim cloth.…”

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confidence: 99%

Semirational Engineering of the Naphthalene Dioxygenase from Pseudomonas sp. NCIB 9816‐4 towards Selective Asymmetric Dihydroxylation

2017

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Enzyme-catalyzed asymmetric dihydroxylation is ap owerful tool fort he selective oxyfunctionalization of various organic compounds. By applying Rieske non-heme dioxygenases (ROs), molecular oxygen and ar eduction equivalent are needed for the generation of vicinal cis-diols. We report ac omprehensive mutagenesis study of the active site of the naphthalene dioxygenasef rom Pseudomonas sp. NCIB 9816-4 comprising 62 variants. We aimed to understand the important structure-function relationships by investigating different substituted arene substrates and the geometry of the active site. Introducing single-point mutations at positions F202, A206, V260, H295, F352, and L307 resulted in drastic shifts in the reactions pecificity,r egioselectivity,a nd stereoselectivity (! 90 %) while maintaining the residual activity towards the natural substrate naphthalene.One of the key reactions in chemistryi st he selective oxyfunctionalization of organic compounds. In this context, chiralv icinal 1,2-diols are of particular interest and are often applied as chiral ligands, auxiliaries, and chiral synthons for pharmaceuticals and agrochemicals.[1] These functionalized compounds are generated by multicomponent Rieske non-heme dioxygenases (ROs), which, analogously to some P450sm onooxygenases, consist of ar eductase, af erredoxin, andahexameric oxygenase.[2] In ROs, categorized as types III and IV,t he reductase obtains electrons from the naturalc osubstrate NAD(P)H,a nd a ferredoxin component shuttles electrons from the reductase to the oxygenase active site, at which actual oxygen activation and substrate hydroxylation occur. [3,4] Representing ab iocatalytic alternative to asymmetricd ihydroxylation with toxic transition-metal catalysts, ROs also exhibit ab road substrate scope.[5] Over 300 diverse substrates ranging from polyaromatic hydrocarbons such as phenanthracene to halogenated ethylenes and flavonoid structures were reportedt ob eh ydroxylated with these enzymes. [6,7] Furthermore, ROs were shown to be suitable for bioremediation applications and to have the ability to process xenobiotics. [8,9] In industry, ROs are utilized to manufacture the blue dye indigo, which is crucial for the production of denim cloth.[10]Oxygen-dependent ROsn ot only convert ah uge variety of compounds, but these catalysts also displayabroad reaction scope. With these enzymes ystems it is possible to address monohydroxylations( Scheme 1, path I), dihydroxylations (Scheme 1, path II), and O-dealkylations (Scheme 1, path III) with high regio-a nd stereoselectivities. [11,12] Recently,s emirational engineering of the cumene dioxygenase (CDO) from Pseudomonas fluorescens IP01 expanded the substrate scope of these versatile catalysts to ar ange of sterically demanding cyclic and linear alkenesa nd monoterpenes. The CDOs ingle variant M232Aw as able to convert different terpeneg eometries such as myrcene and (À)-a-pinene with high specificity and selectivity. [13] This work motivated us to gain furtheri nsighti nto crucial structure-function relation...

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A Versatile High Throughput Screen for Dioxygenase Activity Using Solid-Phase Digital Imaging

Cited by 21 publications

References 19 publications

Laboratory Evolution of Toluene Dioxygenase To Accept 4-Picoline as a Substrate

Laboratory Evolution of Toluene Dioxygenase To Accept 4-Picoline as a Substrate

Probing the Substrate Specificity of Aminopyrrolnitrin Oxygenase (PrnD) by Mutational Analysis

Semirational Engineering of the Naphthalene Dioxygenase from Pseudomonas sp. NCIB 9816‐4 towards Selective Asymmetric Dihydroxylation

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