Identifying the Minimal Enzymes for Unusual Carbon–Sulfur Bond Formation in Thienodolin Biosynthesis

Abstract

Thienodolin (THN) features a tricyclic indole-S-hetero scaffold that encompasses two unique carbon-sulfur bonds. Although its biosynthetic gene cluster has been recently identified in Streptomyces albogriseolus, the essential enzymes for the formation of C-S bonds have been relatively unexplored. Here, we isolated and characterized a new biosynthetic gene cluster from Streptomyces sp. FXJ1.172. Heterologous expression, systematic gene inactivation, and in vitro biochemical characterization enable us to determine the minimum set of genes for THN synthesis, and an aminotransferase (ThnJ) for catalyzing the downstream conversion of tryptophan chlorination. In addition, we evaluated (and mainly excluded) a previously assumed pivotal intermediate by feeding experiments. With these results, we narrowed down four enzymes (ThnC-F) that are responsible for the two unprecedented C-S bond formations. Our study provides a solid basis for further unraveling of the unique C-S mechanisms.

Characterization of the Aminotransferase ThdN from Thienodolin Biosynthesis in Streptomyces albogriseolus

“…[9,15,[19][20][21][22] ThdN is the first aminotransferase to use 6-chloro-l-tryptophan as the natural substrate. [2,3] However,W ang et al reported a K M value of 2.62 mm for ThnJ from the thienodolin producer Streptomyces sp. The highest activity was at pH 8.0;t his is consistentw ith specifications for other tryptophana minotransferases (optimal pH in the slightly alkaline range).…”

“…ThdN is specific for l-tryptophan (not d-tryptophan), as described for other tryptophan aminotransferases. [2] The K M values for the natural substrate (6-chloro-l-tryptophan) and its brominated analogue were found to be significantly lower (66 and 40 mm,r espectively); this is consistent with thienodolin being the major and dechlorothienodolin only am inor product. Thus, the optimal reaction parametersf or its aminating activity were determined for the conversion of 6-chlorotryptophan into 6-chloroindole-3-pyruvic acid.…”

“…The gene for ap otential aminotransferase wasf ound in the thienodolin biosynthetic cluster.D isruption of this gene, thdN,i nt he thienodolin producer S. albogriseolus led to loss of thienodolin formation. [2] Here, we characterized the in vitro aminotransferase activity of ThdN and its involvement in thienodolin biosynthesis. [2] Here, we characterized the in vitro aminotransferase activity of ThdN and its involvement in thienodolin biosynthesis.…”

Development of Halogenase Enzymes for Use in Synthesis

“…141 A number of independent tryptophan 6-halogenases have also been identified recently. [143][144][145][146][147] For example, ThaL from the indole alkaloid thienodolin (29) pathway in Streptomyces albogriseolus is responsible for the 6-chlorination of tryptophan prior to thiophene ring formation. 144,146,148,149 SttH from Streptomyces toxytricini and Th_Hal from the thermophile Streptomyces violaceusniger SPC6 have also been shown to function as tryptophan 6-halogenases.…”

“…[143][144][145][146][147] For example, ThaL from the indole alkaloid thienodolin (29) pathway in Streptomyces albogriseolus is responsible for the 6-chlorination of tryptophan prior to thiophene ring formation. 144,146,148,149 SttH from Streptomyces toxytricini and Th_Hal from the thermophile Streptomyces violaceusniger SPC6 have also been shown to function as tryptophan 6-halogenases. 143,145 Although both have been characterised in vitro and their structures determined, 143,145,150 the biosynthetic pathways in which they function are yet to be explored.…”