Biosynthesis of sibiromycin, a potent antitumor antibiotic

Abstract

Pyrrolobenzodiazepines, a class of natural products produced by actinomycetes, are sequence selective DNA alkylating compounds with significant antitumor properties. Among the pyrrolo[1,4]benzodiazepines (PBDs) sibiromycin, one of two identified glycosylated PBDs, displays the highest affinity for DNA and the most potent antitumor properties. Despite the promising antitumor properties clinical trials of sibiromycin were precluded by the cardiotoxicity effect in animals attributed to the presence of the C-9 hydroxyl group. As a first step toward the development of sibiromycin analogs, we have cloned and localized the sibiromycin gene cluster to a 32.7-kb contiguous DNA region. Cluster boundaries tentatively assigned by comparative genomics were verified by gene replacement experiments. The sibiromycin gene cluster consisting of 26 open reading frames reveals a "modular" strategy in which the synthesis of the anthranilic and dihydropyrrole moieties is completed before assembly by the nonribosomal peptide synthetase enzymes. In addition, the gene cluster identified includes open reading frames encoding enzymes involved in sibirosamine biosynthesis, as well as regulatory and resistance proteins. Gene replacement and chemical complementation studies are reported to support the proposed biosynthetic pathway.

FIG. 1.

(A) Pyrrolobenzodiazepine common ring system. (B) Metabolic precursors and chemical structures of sibiromycin, anthramycin, tomaymycin, and lincomycin A.

FIG. 2.

(A) Genetic organization of the sibiromycin gene cluster. The proposed functions of individual ORFs are summarized in Table 1. (B) HPLC-ESI and bioassay analyses of the secondary metabolites produced by wild-type, ΔsibA, ΔsibE, ΔorfX2, and ΔorfX3 strains.

FIG. 3.

HPLC-ESI and bioassay analyses of the secondary metabolites produced by wild-type and ΔsibC strains, and by chemical complementation of ΔsibC strains with l-kynurenine, 3-hydroxy-l-kynurenine, 3-hydroxyanthranilic acid, and 3-hydroxy-4-methylanthranilic acid.

FIG. 4.

Proposed pathway for the biosynthesis of the 3,5-hydroxy-4-methylanthranilic acid moiety in sibiromycin, which is the suggested substrate for the NRPS enzymes catalyzing diazepine ring formation. Pathway A is favored. The anthramycin biosynthesis is proposed to diverge at the formation of 3-hydroxy-4-methylanthranilic acid.

FIG. 5.

(A) HPLC analyses of the secondary metabolites produced by wild-type and ΔsibG strains. Isolation of the metabolites was carried out by ethyl acetate extractions. (B) ESI spectrum of the HPLC peak eluting at 20.1 min and the chemical structure of the 7-deoxyaglycone of sibiromycin.

FIG. 6.

Proposed pathway for the biosynthesis of the 4-propenyl-2,3-dihydropyrrole-2-carboxylic acid moiety in sibiromycin, which is the suggested substrate for the NRPS enzymes catalyzing diazepine ring formation. The branching points for the anthramycin, tomaymycin, and lincomycin A biosyntheses are indicated.

FIG. 7.

Proposed pathways for the biosynthesis of the sibirosamine moiety with different sequences of the N methylation and glycosyl transfer reactions.