Novel tryptophan metabolism by a potential gene cluster that is widely distributed among actinomycetes

Abstract

The characterization of potential gene clusters is a promising strategy for the identification of novel natural products and the expansion of structural diversity. However, there are often difficulties in identifying potential metabolites because their biosynthetic genes are either silenced or expressed only at a low level. Here, we report the identification of a novel metabolite that is synthesized by a potential gene cluster containing an indole prenyltransferase gene (SCO7467) and a flavin-dependent monooxygenase (FMO) gene (SCO7468), which were mined from the genome of Streptomyces coelicolor A3(2). We introduced these two genes into the closely related Streptomyces lividans TK23 and analyzed the culture broths of the transformants. This process allowed us to identify a novel metabolite, 5-dimethylallylindole-3-acetonitrile (5-DMAIAN) that was overproduced in the transformant. Biochemical characterization of the recombinant SCO7467 and SCO7468 demonstrated the novel L-tryptophan metabolism leading to 5-DMAIAN. SCO7467 catalyzes the prenylation of L-tryptophan to form 5-dimethylallyl-L-tryptophan (5-DMAT). This enzyme is the first actinomycetes prenyltransferase known to catalyze the addition of a dimethylallyl group to the C-5 of tryptophan. SCO7468 then catalyzes the conversion of 5-DMAT into 5-dimethylallylindole-3-acetaldoxime (5-DMAIAOx). An aldoxime-forming reaction catalyzed by the FMO enzyme was also identified for the first time in this study. Finally, dehydration of 5-DMAIAOx presumably occurs to yield 5-DMAIAN. This study provides insight into the biosynthesis of prenylated indoles that have been purified from actinomycetes.

FIGURE 1.

Gene clusters containing the indole prenyltransferase gene. The Type A gene clusters contain tryptophanase genes. This type of cluster is associated with the biosynthesis of 6-dimethylallylindole-3-carbardehyde. The Type B gene clusters contain flavin-dependent monooxygenase genes. The function of the Type B clusters has not been identified.

FIGURE 2.

Heterologous expression of SCO7467 and SCO7468 in S. lividans TK23. A, plasmids used for heterologous expression. B, HPLC analysis of the metabolites of the S. lividans TK23 transformants harboring pSE101, pSCO101, pSCO102, or pSCO103. S. lividans TK23/pSE101 was used as a control. C, LC-MS/MS fragmentation patterns of authentic IAN and metabolites from S. lividans TK23/pSCO103 and S. lividans TK23/pSCO102. D, structures of IAN (1) and 5-DMAIAN (2).

FIGURE 3.

Characterization of recombinant SCO7467 and SCO7468. A, SDS-PAGE of the recombinant SCO7467. B, SDS-PAGE of the recombinant SCO7468. C, HPLC analysis of SCO7468-bound flavin cofactor. D, UV-visible spectra of flavin co-factors in each sample.

FIGURE 4.

Prenyltransferase activity of recombinant SCO7467. A, in vitro analysis of the SCO7467 reaction with l-tryptophan (3). SCO7467 efficiently converted 3 to 5-DMAT (4) over a 10-min incubation. B, in vitro analysis of the SCO7467 reaction with 1. SCO7467 catalyzed the prenylation of 1 to give a small amount of 2-DMAIAN (5) and 6-DMAIAN (6). C, steady-state kinetics of SCO7467. Michaelis-Menten plots of the SCO7467-catalyzed reactions using various concentrations of l-tryptophan (left) and DMAPP (right) are shown. The concentration of DMAPP or l-tryptophan was fixed at 1 mm when the concentration of the other substrate varied.

FIGURE 5.

In vitro analysis of the SCO7468 reaction. A, in vitro analysis of the SCO7468 reaction with 5-DMAT (4). SCO7468 completely converted 4 to 5-DMAIAOx (7) in an NADPH-dependent manner over a 10-min incubation. B, in vitro analysis of the SCO7468 reaction with 3. The 10-min incubation of SCO7468 resulted in almost no substrate depletion and almost no product formation. In contrast, the prolonged 2-h incubation with 1 mm 3 resulted in the formation of IAOx (8). C, LC-MS/MS fragment patterns of authentic IAOx and SCO7468 reaction products detected in the chromatograms B. D, steady-state kinetics of SCO7468. Michaelis-Menten plots of the SCO7468-catalyzed reactions using various concentrations of 5-DMAT (left) and l-tryptophan (right) are shown. See “Experimental Procedures” for details.

FIGURE 6.

In vitro analysis of the SCO7468 reaction using N-hydroxytryptophan as a substrate. A, extracted ion current chromatograms at m/z 221.0921 ± 0.0005 for N-hydroxytryptophan (9). Reaction mixtures: a, with SCO7468 for 60 min; b, without SCO7468 for 60 min; c, 0 min (control). N-Hydroxytryptophan (9) was completely consumed in a 60-min incubation with SCO7468 (a). B, extracted ion current chromatograms at m/z 175.0866 ± 0.0005 for IAOx (8). Reaction mixtures: a, with SCO7468 for 60 min; b, without SCO7468 for 60 min; c, 0 min (control). IAOx (8) was fully produced in a 60-min incubation with SCO7468 (a). Nonenzymatic formation of 8 was also detected at a trace level (b and c). C, proposed reaction mechanism for the SCO7468-catalyzed formation of 8 from 3 via 9. N,N-Dihydroxytryptophan and 3-(1H-indol-3-yl)-2-nitrosopropanic acid are deduced intermediates of the SCO7468 reaction.

FIGURE 7.

Proposed pathway for prenylated indole biosynthesis. A, l-tryptophan metabolism in actinomycetes by the Type A gene cluster. B, l-tryptophan metabolism in actinomycetes by the Type B gene cluster. The reactions demonstrated in this study are framed.