学位论文详细信息
Biosynthesis of the thiopetins and identification of an F420H2-dependent dehydropiperidine reductase
Biosynthesis thiopeptide
Ichikawa, Hiroyuki ; Kelly, Wendy L. Chemistry and Biochemistry Bommarius, Andreas S. Kubanek, Julia M. Lobachev, Kirill S. Oyelere, Adegboyega K. ; Kelly, Wendy L.
University:Georgia Institute of Technology
Department:Chemistry and Biochemistry
关键词: Biosynthesis thiopeptide;   
Others  :  https://smartech.gatech.edu/bitstream/1853/62709/1/ICHIKAWA-DISSERTATION-2019.pdf
美国|英语
来源: SMARTech Repository
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【 摘 要 】

Thiopeptins are highly decorated thiopeptide antibiotics similar in structure to thiostrepton A and harbor two unusual features. All thiopeptins contain a thioamide, a rare moiety among natural products, and a subset of thiopeptins present with a piperidine in the core macrocycle rather than the more oxidated dehydropiperidine or pyridine rings typically observed in the thiopeptides. Here, we report the identification of the thiopeptin biosynthetic gene (tpn) cluster in Streptomyces tateyamensis and the gene products, TpnLMNW. TpnW shows sequence similarity to transaminases and is likely involved in the biosynthesis of the 4-(1-hydroxyethyl)quinoline-2-carboxylic acid (HEQ) moiety of thiopeptin. HEQ is derived from L-tryptophan and the second step of HEQ biosynthesis requires a transaminase that catalyzes the conversion of 2-methyl-L-tryptophan to 3-(2-methylindolyl)pyruvate. In vitro reconstitution of TpnW activity confirmed that this enzyme is a 2-methyl-L-tryptophan aminotransferase. TpnW is able to utilize 3 indolylpyruvate, p-hydroxyphenylpyruvate, and phenylpyruvate as α-keto acid acceptors but not pyruvate, oxaloacetate, or α-ketoglutarate. TpnMN share homology to YcaO and TfuA proteins, respectively, responsible for thioamidation of peptidic substrates. Heterologous expression of TpnMN in the thiostrepton A producer, Streptomyces laurentii (S. laurentii), led to the production of a metabolite with a mass matching a thioamidated thiostrepton A. Structural characterization of this metabolite is required to determine if it contains a thioamide suggested by the proposed functions of TpnMN. TpnL shows sequence similarity to (deaza)flavin-dependent oxidoreductases. Heterologous expression of TpnL in the thiostrepton A producer, S. laurentii, led to the production of a piperidine-containing analog. Binding studies revealed TpnL preferentially binds the deazaflavin cofactor, coenzyme F420, and in vitro reconstitution of TpnL activity confirmed that this enzyme is an F420H2-dependent dehydropiperidine reductase. The identification of TpnL and its activity establishes the basis for the piperidine-containing series a thiopeptides, one of the five main structural groups of this diverse family of antibiotics. This work was published in J. Am. Chem. Soc. in 2018.

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