Protein engineering of toluene ortho-monooxygenase of Burkholderia cepacia G4 for regiospecific hydroxylation of indole to form various indigoid compounds

Abstract

Previous work showed that random mutagenesis produced a mutant of toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 containing the V106A substitution in the hydroxylase alpha-subunit (TomA3) that changed the color of the cell suspension from wild-type brown to green in rich medium. Here, DNA shuffling was used to isolate a random TOM mutant that turned blue due to mutation TomA3 A113V. To better understand the TOM reaction mechanism, we studied the specificity of indole hydroxylation using a spectrum of colored TOM mutants expressed in Escherichia coli TG1 and formed as a result of saturation mutagenesis at TomA3 positions A113 and V106. Colonies expressing these altered enzymes ranged in color from blue through green and purple to orange; and the enzyme products were identified using thin-layer chromatography, high performance liquid chromatography, and liquid chromatography-mass spectroscopy. Derived from the single TOM template, enzymes were identified that produced primarily isoindigo (wild-type TOM), indigo (A113V), indirubin (A113I), and isatin (A113H and V106A/A113G). The discovery that wild-type TOM formed isoindigo via C-2 hydroxylation of the indole pyrrole ring makes this the first oxygenase shown to form this compound. Variant TOM A113G was unable to form indigo, indirubin, or isoindigo (did not hydroxylate the indole pyrrole ring), but produced 4-hydroxyindole and unknown yellow compounds from C-4 hydroxylation of the indole benzene ring. Mutations at V106 in addition to A113G restored C-3 indole oxidation, so along with C-2 indole oxidation, isatin, indigo, and indirubin were formed. Other TomA3 V106/A113 mutants with hydrophobic, polar, or charged amino acids in place of the Val and/or Ala residues hydroxylated indole at the C-3 and C-2 positions, forming isatin, indigo, and indirubin in a variety of distributions. Hence, for the first time, a single enzyme was genetically modified to produce a wide range of colors from indole.

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

“…1A) (13,31). Similarly, recent studies with the toluene-2-monooxygenase (T2MO) of Burkholderia cepacia G4 indicate that protein engineering of the diironcontaining monooxygenase can alter the ratio of the indolederived products produced (4-hydroxyindole, isatin, indigo, indirubin, and isoindigo), which in part accounts for the range of colors seen in the chloroform extracts of cultures expressing the modified isoforms of T2MO (38).…”

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.…”

Alpha-Subunit Positions Methionine 180 and Glutamate 214 of Pseudomonas stutzeri OX1 Toluene- o -Xylene Monooxygenase Influence Catalysis

“…Structure-function relationships are beginning to be understood to the extent that it is now possible to hydroxylate the benzene ring of toluene at all possible positions (4). Through DNA shuffling of these oxygenases, alpha-subunit positions I100 (2), A107 (11), E214G/D312N/M399V (19), and M180T/ E284G (19,20) have been identified which influence catalysis. Here, saturation mutagenesis of the alpha-subunit (TouA) of toluene-o-xylene monooxygenase (ToMO) showed that TouA M180 influences the regiospecificity of hydroxylation of substituted aromatics and TouA E214 influences the catalysis rate.…”

“…Position E214 influences the rate of reaction (e.g., E214G increases p-nitrophenol oxidation 15-fold) by controlling substrate entrance and product efflux as a gate residue.Toluene monooxygenases (1,5,7,15,21) are multisubunit catalysts that oxidize benzene to phenol, catechol, and trihydroxybenzene (18,20) and may be engineered to produce a range of methyl-, nitro-, and methoxy-substituted aromatics with industrial and pharmaceutical value (2,3,11,16,17,19,20). Structure-function relationships are beginning to be understood to the extent that it is now possible to hydroxylate the benzene ring of toluene at all possible positions (4).…”