Mutations of Toluene-4-Monooxygenase That Alter Regiospecificity of Indole Oxidation and Lead to Production of Novel Indigoid Pigments

Abstract

Broad-substrate-range monooygenase enzymes, including toluene-4-monooxygenase (T4MO), can catalyze the oxidation of indole. The indole oxidation products can then condense to form the industrially important dye indigo. Site-directed mutagenesis of T4MO resulted in the creation of T4MO isoforms with altered pigment production phenotypes. High-pressure liquid chromatography, thin-layer chromatography, and nuclear magnetic resonance analysis of the indole oxidation products generated by the mutant T4MO isoforms revealed that the phenotypic differences were primarily due to changes in the regiospecificity of indole oxidation. Most of the mutations described in this study changed the ratio of the primary indole oxidation products formed (indoxyl, 2-oxindole, and isatin), but some mutations, particularly those involving amino acid G103 of tmoA, allowed for the formation of additional products, including 7-hydroxyindole and novel indigoid pigments. For example, mutant G103L converted 17% of added indole to 7-hydroxyindole and 29% to indigoid pigments including indigo and indirubin and two other structurally related pigments. The double mutant G103L:A107G converted 47% of indole to 7-hydroxyindole, but no detectable indigoid pigments were formed, similar to the product distribution observed with the toluene-2-monooxygenase (T2MO) of Burkholderia cepacia G4. These results demonstrate that modification of the tmoA active site can change the products produced by the enzyme and lead to the production of novel pigments and other indole oxidation products with potential commercial and medicinal utility.

SmoXYB1C1Z of Mycobacterium sp. Strain NBB4: a Soluble Methane Monooxygenase (sMMO)-Like Enzyme, Active on C 2 to C 4 Alkanes and Alkenes

“…No epoxide was detected in cell suspensions of mc 2 -155(pMycoFos) (data not shown). The mc 2 -155(pSmo) cells did not react visibly with indole; this gives indigoid pigments in the case of many oxygenases (40)(41)(42).…”

Isolation of Indole Utilizing Bacteria Arthrobacter sp. and Alcaligenes sp. From Livestock Waste

“…Various reports show that the hydroxylated and oxygenated products of indole degradation, such as, isatin, indoxyl, 7-hydroxyindole, 2-hydroxyindole, and 3-oxindole, which are used in the synthesis of indigo dyes by dimerization [28,29]. Thus, those bacteria that are known to transform indole into hydroxylated indole intermediates might produce dimers.…”

“…Thus, those bacteria that are known to transform indole into hydroxylated indole intermediates might produce dimers. Recombinant technology has resulted in the cloning of some known or putative aromatic monooxygenases or dioxygenases in E. coli, and greatly enhanced indigo formation [28][29][30][31]. The accumulation of indigo as a dimer product during the indole degradation is the disadvantage for the application of those microorganisms in the in situ attenuation of indole odor emitted from different types of waste.…”

Phenotypic and physiological alterations by heterologous acylhomoserine lactone synthase expression in Pseudomonas putida

“…PpoR also appears likely to repress indole degradation, which could be released in the presence of the HHL signal. It is worth investigating whether the pink pigment observed is indirubin because of its anti-cancer effects (McClay et al, 2005).…”

“…Indole is oxidized by a number of oxygenase enzymes, resulting in the production of indigo (blue pigment) and the related indigoid pigment, indirubin (pink pigment) (McClay et al, 2005). In order to determine whether the pigment was the product of indole degradation, we measured indole degradation via HPLC analysis.…”