Cetoniacytone A

Cetoniacytone A (1) and some related minor components (2, 6, 7) were produced by Actinomyces sp. (strain Lu 9419), which was isolated from the intestines of a rose chafer (Cetonia aureata). The structures of the novel metabolites were established by detailed spectroscopic analysis. The absolute configuration of 1 was determined by X-ray analysis and derivatisation with chiral acids. 1 exhibits a significant cytotoxicity against selected tumor cell lines. The biosynthesis of 1 was studied by feeding 13C labelled precursors. The results suggest that the characteristic p-C7N skeleton of the aminocarba sugar is formed via the pentose phosphate pathway by cyclisation of a heptulose phosphate intermediate.

Sugar phosphate cyclases (SPCs) catalyze the cyclization of sugar phosphates to produce a variety of cyclitol intermediates that serve as the building blocks of many primary metabolites, for example, aromatic amino acids, and clinically relevant secondary metabolites, for example, aminocyclitol/aminoglycoside and ansamycin antibiotics. Feeding experiments with isotopically labeled cyclitols revealed that cetoniacytone A, a unique C(7)N-aminocyclitol antibiotic isolated from an insect endophytic Actinomyces sp., is derived from 2-epi-5-epi-valiolone, a product of SPC. By using heterologous probes from the 2-epi-5-epi-valiolone synthase class of SPCs, an SPC homologue gene, cetA, was isolated from the cetoniacytone producer. cetA is closely related to BE-orf9 found in the BE-40644 biosynthetic gene cluster from Actinoplanes sp. strain A40644. Recombinant expression of cetA and BE-orf9 and biochemical characterization of the gene products confirmed their function as 2-epi-5-epi-valiolone synthases. Further phylogenetic analysis of SPC sequences revealed a new clade of SPCs that might regulate the biosynthesis of a novel set of secondary metabolites.

A gene cluster responsible for the biosynthesis of the antitumor agent cetoniacytone A was identified in Actinomyces sp. strain Lu 9419, an endosymbiotic bacterium isolated from the intestines of the rose chafer beetle (Cetonia aurata). The nucleotide sequence analysis of the 46 kb DNA region revealed the presence of 31 complete ORFs, including genes predicted to encode a 2-epi-5-epi-valiolone synthase (CetA), a glyoxalase/bleomycin resistance protein (CetB), an acyltransferase (CetD), an FAD-dependent dehydrogenase (CetF2), two oxidoreductases (CetF1 and CetG), two aminotransferases (CetH and CetM), and a pyranose oxidase (CetL). CetA has previously been demonstrated to catalyze the cyclization of sedoheptulose 7-phosphate to the cyclic intermediate, 2-epi-5-epi-valiolone. In this report, the glyoxalase/bleomycin resistance protein homolog CetB was identified as a 2-epi-5-epi-valiolone epimerase (EVE), a new member of the vicinal oxygen chelate (VOC) superfamily. The 24 kDa recombinant histidine-tagged CetB was found to form a homodimer; each monomer contains two betaalphabetabetabeta scaffolds that form a metal binding site with two histidine and two glutamic acid residues. A BLAST search using the newly isolated cet biosynthetic genes revealed an analogous suite of genes in the genome of Frankia alni ACN14a, suggesting that this plant symbiotic nitrogen-fixing bacterium is capable of producing a secondary metabolite related to the cetoniacytones.