Investigation of the Unusual Carbon-Carbon Bond Formation in Cylindrocyclophane Biosynthesis

抄録

Nature constructs structurally diverse, bioactive molecules using enzymes. Many enzymes catalyze synthetically challenging reactions under mild, physiological conditions. Consequently, they have long been a source of inspiration for developing biomimetic organic syntheses and methods. In addition, enzymes are increasingly being used as biocatalysts in industry. Therefore, the discovery of enzymes that catalyze chemically intriguing transformations can positively impact synthesis in multiple ways. With the recent advances in next-generation DNA sequencing technologies, we are now able to access enormous amount of genomic sequencing data, which encodes a treasure chest of new enzymatic chemistry. The challenge now is to devise a method to efficiently identify chemically interesting enzymes from this vast pool of information.

One possible solution to this problem is to study the biosynthetic pathways of structurally unique natural products, which are predicted to involve novel enzymatic reactions. We aimed to discover new enzymes that catalyze intriguing chemical reactions through biosynthetic investigation guided by our knowledge in organic chemistry. The cylindrocyclophanes are a family of natural products that contain an unusual [7.7]paracyclophane core scaffold.¹ Based on the results of the previous feeding studies, cylindrocyclophane biosynthesis is predicted to involve an unusual C–C bond formation (Figure 1).² To discover the enzymes responsible for this chemistry, we studied the biosynthesis of the cylindrocyclophanes.

Figure 1. The structures of the cylindrocyclophanes. The predicted biosynthetic disconnection suggests that an unusual C–C bond formation is involved in their biosynthesis.

First, the candidate cylindrocyclophane biosynthetic (cyl) gene cluster was identified from the genomic sequence of the cylindrocyclophane producer, Cylindrospermum licheniforme ATCC 29412. We next formulated a biosynthetic hypothesis based on the cyl gene cluster annotation (Figure 2). In our original biosynthetic hypothesis, we predicted that cylindrocyclophane biosynthesis initiates with the activation of decanoic acid by the fatty acid activating enzymes, CylA and CylB, to form decanoyl-CylB. The activated decanoyl-CylB is then processed by the type I polyketide synthase (PKS) machinery, CylD-H. The nascent polyketide is released from the type I PKS assembly line by the type III PKS CylI to form the alkylresorcinol, which is the predicted monomeric unit of the cylindrocyclophanes.

Figure 2. The initial biosynthetic hypothesis for cylindrocyclophane assembly.

Based on our initial biosynthetic hypothesis, we biochemically characterized the functions of the fatty acid activating enzymes CylA/CylB and the type III PKS CylI. The in vitro activities of these three enzymes were consistent with our biosynthetic hypothesis, which validated the involvement of the cyl gene cluster in cylindrocyclophane production.³ In addition, we conducted feeding experiments using deuterium-labeled decanoic acid in the native producer to confirm that decanoic acid is a precursor to the cylindrocyclophanes. The incorporation of deuterium-labeled decanoic acid into the final cylindrocyclophane scaffold also indicated that cylindrocyclophane biosynthesis involves functionalization of the unactivated carbon center.³

Following our discovery and validation of the cyl gene cluster, we next focused on the investigation of the key C–C bond formation that results in the construction of the [7.7]paracyclophane scaffold. Through bioinformatics and biochemical characterizations of the enzymes encoded in the cyl gene cluster, we determined that cylindrocyclophane biosynthesis involv

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