Identification of (2S,3S)-β-Methyltryptophan as the Real Biosynthetic Intermediate of Antitumor Agent Streptonigrin

Abstract

Streptonigrin is a potent antitumor antibiotic, active against a wide range of mammalian tumor cells. It was reported that its biosynthesis relies on (2S,3R)-β-methyltryptophan as an intermediate. In this study, the biosynthesis of (2S,3R)-β-methyltryptophan and its isomer (2S,3S)-β-methyltryptophan by enzymes from the streptonigrin biosynthetic pathway is demonstrated. StnR is a pyridoxal 5′-phosphate (PLP)-dependent aminotransferase that catalyzes a transamination between L-tryptophan and β-methyl indolepyruvate. StnQ1 is an S-adenosylmethionine (SAM)-dependent C-methyltransferase and catalyzes β-methylation of indolepyruvate to generate (R)-β-methyl indolepyruvate. Although StnR exhibited a significant preference for (S)-β-methyl indolepyruvate over the (R)-epimer, StnQ1 and StnR together catalyze (2S,3R)-β-methyltryptophan formation from L-tryptophan. StnK3 is a cupin superfamily protein responsible for conversion of (R)-β-methyl indolepyruvate to its (S)-epimer and enables (2S,3S)-β-methyltryptophan biosynthesis from L-tryptophan when combined with StnQ1 and StnR. Most importantly, (2S,3S)-β-methyltryptophan was established as the biosynthetic intermediate of the streptonigrin pathway by feeding experiments with a knockout mutant, contradicting the previous proposal that stated (2S,3R)-β-methyltryptophan as the intermediate. These data set the stage for the complete elucidation of the streptonigrin biosynthetic pathway, which would unlock the potential of creating new streptonigrin analogues by genetic manipulation of the biosynthetic machinery.

Xantholipin B produced by the stnR inactivation mutant Streptomyces flocculus CGMCC 4.1223 WJN-1

“…Subsequently, StnK3 catalyzes epimerization, and StnR catalyzes a second round of transamination to complete the assembly of (2S,3S)-β-methyltryptophan. 22,23 To confirm the role of stnR in streptonigrin biosynthesis, stnR was inactivated in vivo by replacing an internal fragment of stnR with the apramycin resistance cassette to generate WJN-1, and the genotype was verified by PCR (Supplementary Figure S1). To evaluate the effect of stnR replacement, the production of streptonigrin in WJN-1 was monitored by HPLC and LC-MS. Streptonigrin production was completely abolished in the strain WJN-1 and, surprisingly, two new compounds (1 and 2), absent in the wild-type strain, were present in the mutant strain WJN-1 ( Figure 2 and Supplementary Figure S16).…”

Identification of the Antifungal Metabolite Chaetoglobosin P From Discosia rubi Using a Cryptococcus neoformans Inhibition Assay: Insights Into Mode of Action and Biosynthesis

“…(2S,3S)-β-methyltryptophan is found in metabolites such as, maremycins, Fr99452, lavendamycin, and streptonigrin ( Liu B. et al, 2019 ), and (2S,3R)-β-MeTrp is incorporated into indolmycin and telomycin. Formation of the (2S,3S)-β-methyltryptophan biosynthetic precursor of streptonigrin ( Xu et al, 2013 ; Kong et al, 2016 ) is catalyzed by enzymes encoded by the stnR-stnQ1-stnK3 cassette. Similarly, the marG-marH-marI gene cassette of the maremycin pathway from Streptomyces sp.…”

Biosynthetic study of β-methyl amino acid building blocks involved in natural products

“…框组成的基因盒, stnQ1-stnK3-stnR, 分别编码甲基转 移酶、异构酶和氨基转移酶. Kong等人 [37] 对stnQ1进 D2 [38,39] , 以及含有螺环共轭氧吲哚单元的Maremycin E, F和G [39,40] . [43,44] .…”