Characterization of Yatakemycin Gene Cluster Revealing a Radical S-Adenosylmethionine Dependent Methyltransferase and Highlighting Spirocyclopropane Biosynthesis

Abstract

Yatakemycin (YTM), an antitumor natural product, represents the most potent member of a class of potent anticancer natural products including CC-1065 and duocarmycins. Herein we describe the biosynthetic gene cluster of YTM, which was identified by genome scanning of Streptomyces sp. TP-A0356. This cluster consists of 31 open reading frames (ORFs) and was localized to a 36 kb DNA segment. Moreover, its involvement in YTM biosynthesis was confirmed by cluster deletion, gene replacement, and complementation. Inactivation of ytkT, which encodes a radical S-adenosylmethionine (SAM) protein, created a mutant strain that failed to produce YTM but accumulated a new metabolite, which was structurally elucidated as a precursor that was related to the formation of the cyclopropane ring. More importantly, biochemical characterization of the radical SAM-dependent enzyme YtkT revealed that it is a novel C-methyltransferase and contributes to an advanced intermediate during formation of the cyclopropane ring through a radical mechanism in the YTM biosynthetic pathway. On the basis of in silico analysis, genetic experiments, structure elucidation of the novel intermediate, and biochemical characterization, a biosynthetic pathway for yatakemycin was proposed, which sets the stage to further investigate the novel enzymatic mechanisms and engineer the biosynthetic machinery for the production of novel analogues.

Radical new paradigm for heme degradation in Escherichia coli O157:H7

“…Clues to the ChuW mechanism come from what has been recently observed for other class C RSMTs. All known class C RSMTs catalyze methylations of sp 2 -hybridized carbon atoms that, in some cases, lead to considerable structural rearrangement (22,23,31,32). Although ChuW/HutW's appear to be evolutionarily distinct (Fig.…”

“…Of particular interest are the products of YtkT and Jaw5 that have been shown to contain other "high energy" C1-derived moieties, such as cyclopropane rings (22,31). Such a moiety may be unstable if placed across the 4-5 or 5-6 carbon-carbon bond of deuteroheme (Fig.…”

“…methyl groups to unreactive carbon atoms and, in some cases, promote significant chemical rearrangements (21,22). However, they are the least understood of the four distinguished classes (23), making it difficult to determine function or mechanism using only a bioinformatics approach.…”

Mining of a streptothricin gene cluster from Streptomyces sp. TP-A0356 genome via heterologous expression

“…TP-A0356 was described previously [23], and the sequence of the ST cluster is available in GenBank under accession number KC935381. FramePlot 4.0 beta (http://nocardia.nih.go.jp/ fp4) was used to deduce possible open reading frames (ORFs).…”

“…TP-A0356 genome was described previously [23]. The cosmids containing putative genes responsible for the biosynthesis of streptothricins were screened by PCR using primers stnD-For (5′-CGACAGCGGCTGGGACTTCC-3′) and stnD-Rev (5′-ACCCAGATGAAGGACGCCCG-3′), stnO-For (5′-CTGG-ACGAGGGTTTCTACGG-3′) and stnO-Rev (5′-GCACA-GGTCGAGCAGGTGG-3′).…”