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.

Enterotoxicity of a nonribosomal peptide causes antibiotic-associated colitis

“…The K. oxytoca PAI genes show >50% amino acid sequence similarity to PBD synthesis genes from soil bacteria belonging to Actinomycetales, Streptomyces and Streptosporangium (Fig. S2B), which produce low-molecular-weight effector molecules with potent cytostatic activities (Fig.1E) (33)(34)(35). A capacity for production of other metabolites or virulence factors is not apparent within the PAI genes (Table S1).…”

Cloning and Characterization of the Biosynthetic Gene Cluster for Tomaymycin, an SJG-136 Monomeric Analog

“…The terminal carbon of the propylidene side chain shown by labeling experiments to derive by L-methionine is likely added by an S-adenosylmethionine-dependent methyltransferase. In the accompanying study (18) we propose that SibZ from the sibiromycin gene cluster catalyzes a methyl transfer coupled to tautomerization. This assignment is further confirmed by the absence of a homologous enzyme in the tomaymycin gene cluster, which does not require this activity.…”

“…The first two enzymes are similar to the tyrosine hydroxylase and L-DOPA-2,3-dioxygenase of the lincomycin A biosynthesis (24, 25) (Table 1). A BLAST search of TomK shows no similar enzyme except for LmbX, ORF15, and SibS ( Table 1) that was proposed to catalyze the unusual C-C hydrolysis of 4-(3-carboxy-3-oxo-propenyl)-2,3-dihydropyrrole-2-carboxylic acid (8,18).…”

“…Gene inactivation experiments, bioassays, and HPLC-ESI analyses confirmed this assignment. Upstream of orfX, tomQ encodes a putative flavin-dependent oxidoreductase with high similarity with ORF24 of the anthramycin gene cluster (see the accompanying study [18] and Table 1). Gene inactivation of tomQ did not abolish tomaymycin production, as judged by HPLC-ESI analysis, suggesting that this enzyme is not essential for tomaymycin biosynthesis.…”

“…The first for the biosynthesis of PBDs with a C-9 hydroxyl group such as anthramycin and sibiromycin ( Fig. 1) has adopted the L-tryptophan degradation biosynthetic machinery from primary metabolism for the formation of the intermediate 3-hydroxyanthranilic acid whose hydroxyl group ends up at C-9 in the PBD ring system (18). The second is used for the biosynthesis of PBDs not substituted at C-9, such as tomaymycin (Fig.…”