Unified Biosynthetic Origin of the Benzodipyrrole Subunits in CC-1065

Abstract

CC-1065 is the first characterized member of a family of naturally occurring antibiotics including yatakemycin and duocarmycins with exceptionally potent antitumor activity. CC-1065 contains three benzodipyrroles (1a-, 1b-, and 1c-) of which the 1a-subunit is remarkable by being composed of a cyclopropane ring, and the mechanism for the biological formation of benzodipyrrole rings remains elusive. Previously, biosynthetic studies of CC-1065 were limited to radioactively labeled precursor feeding experiments, which showed that tyrosine (Tyr) and serine (Ser) were incorporated into the two benzodipyrrole (1b- and 1c-) subunits via the same mode but that this was different from the key cyclopropabenzodipyrrole (1a-) subunit with N1-C2-C3 derived from Ser. Herein, the biosynthetic gene cluster of CC-1065 has been cloned, analyzed, and characterized by a series of gene inactivations. Significantly, a key intermediate bearing a C7-OH group derived from a Δc10C mutant exhibited improved cytotoxicity. Moreover, this data inspired us to suspect that the 1a-subunit might employ the same precursor incorporation mode as the 1b- and 1c-subunits. Subsequently, C-labeled Tyr feeding experiments confirmed that the N1-C2-C3 is originated from Tyr via DOPA as an intermediate. Collectively, a biosynthetic pathway of benzodipyrrole is proposed featuring a revised and unified precursor incorporation mode, which implicates an oxidative cyclization strategy for the assembly of benzodipyrrole. This work sets the stage for further study of enzymatic mechanisms and combinatorial biosynthesis for new DNA alkylating analogues.

A widely distributed metalloenzyme class enables gut microbial metabolism of host- and diet-derived catechols

“…These organisms come 425 from mammal-associated, plant-associated, soil, and aquatic habitats. Notable organisms encoding 426 putative dehydroxylases include soil-dwelling Streptomycetes (49,50), the industrially important 427…”

“…For instance, two catechol dehydroxylase homologs from Streptomyces 441 are present in biosynthetic gene clusters that produce the potent anti-tumor compounds 442 yatakemycin and CC-1065 (49,50) ( Fig 5B). Gene knock-out and complementation studies 443 revealed that the dehydroxylase homolog is essential for CC-1065 production and likely catalyzes 444 reductive dehydroxylation of a late-stage biosynthetic intermediate (49). Another homolog is 445 present in the 3,5-dihydroxybenzoate (3,5-DHB) degradation operon within the anaerobic soil 446…”

A radical S-adenosyl-L-methionine enzyme and a methyltransferase catalyze cyclopropane formation in natural product biosynthesis

“…Our group has focused on the biosynthetic studies of 1 and 3 , and we have identified their biosynthetic gene clusters (BGCs), unraveled a unified biosynthetic origin of the benzodipyrrole subunits in 1 , and proposed their biosynthetic pathways, respectively 29 , 30 . Gene inactivation and biosynthetic intermediate characterization confirmed that a HemN-like radical SAM enzyme C10P was involved in the formation of the cyclopropane moiety in 1 29 , but how the cyclopropane formation proceeds is still obscure. Here, we show that a methyltransferase C10Q is also required for the cyclopropanation process.…”

GyrI-like proteins catalyze cyclopropanoid hydrolysis to confer cellular protection

“…1b , iv). Given the fact that C10R6 is encoded by the biosynthetic gene cluster of 2 21 , this protein may function to catalyze CC-1065 cyclopropyl hydrolysis. Indeed, incubation of C10R6 with 2 showed that C10R6 could convert 2 to a new derivative 7 (Fig.…”