Functional analysis of the biosynthetic gene cluster of the antitumor agent cetoniacytone A Public Deposited

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  • A gene cluster responsible for the biosynthesis of the antitumor agent cetoniacytone A was identified in Actinomyces sp. strain Lu 9419, an endosymbiotic bacteria isolated from the intestines of the rose chafer beetle. The nucleotide sequence analysis of the 26 Kb DNA region revealed the presence of 17 complete ORFs, including genes predicted to encode a 2-epi-5-epi-valiolone synthase (CetA), a glyoxalase (CetB), an FAD/FMN-dependent dehydrogenase (CetF), an oxidoreductase (CetG), two aminotransferases (CetH, CetM), and a pyranose oxidase (CetL). BLAST search analysis using the newly isolated cet biosynthetic pathway revealed a homologous biosynthetic pathway in the genome of Frankia alni ACN14a, suggesting that this organism is capable of producing a metabolite related to the cetoniacytones. The 2-epi-5-epi-valiolone synthase (CetA) was cloned and expressed in E. coli and biochemical characterization of the gene product revealed that CetA is capable of catalyzing the cyclization of sedoheptulose 7-phosphate to 2-epi-5-epivaliolone. In addition, three other 2-epi-5-epi-valiolone synthase genes, from different natural product biosynthetic pathways have also been recombinantly expressed and biochemically characterized. These include BE-orf9 from the BE- 40644 biosynthetic gene cluster, prlA from the pyralomicin biosynthetic gene cluster, and salQ from the salbostatin biosynthetic gene cluster. Comparative analysis of the gene products with other related cyclases that are involved in natural product biosynthesis revealed that the 2-epi-5-epi-valiolone synthases uniquely represent a class of sugar phosphate cyclases (SPCs) that has a catalytic mechanism similar to that of dehydroquinate synthase in the shikimate pathway. Enzymes that belong to the SPC superfamily catalyze the cyclization of sugar phosphates to produce a variety of cyclitol intermediates that serve as the building blocks of many primary and secondary metabolites. Further phylogenetic analysis of SPC sequences revealed a new clade of SPCs, consisting a group of proteins with unknown function, that may regulate the biosynthesis of a novel set of secondary metabolites. The product of cetB, which has high identity to glyoxalases (members of the vicinal oxygen chelate (VOC) superfamily), has also been characterized. Members of the VOC superfamily catalyze a large range of divalent metal ion-dependent reactions. Enzymatic characterization of CetB revealed that this enzyme was able to catalyze the second metabolic step in cetoniacytone biosynthesis, mediating the epimerization of 2-epi-5-epi-valiolone to 5-epi-valiolone. Therefore, CetB may be designated as a new member of the VOC superfamily. Site directed nutagenesis and metal binding analysis showed that CetB is a Ni²⁺-dependent protein with four metal binding sites. Similar to other members of the VOC superfamily, CetB contains the common structural βαβββ scaffold, as predicted through Phyre program. Native protein gel and size exclusion analyses have shown that CetB exists as a two-module protein dimer. The results provide important insight into the mode of formation of this unique aminocyclitol natural product, and will contribute to future studies that aim to create new aminocyclitol analogs.
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