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Functional characterization of Gra-6 as a branch-point enzyme directing (R)-DNPA formation in BIQ polyketide biosynthesis

The Journal of Antibiotics (2026)Cite this article

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Abstract

Benzoisochromanequinone (BIQ) polyketides including actinorhodin, medermycin, and granaticin, are biosynthesized via the intermediate 4-dihydro-9-hydroxy-1-methyl-10-oxo-3-H-naphtho[2,3-c]pyran-3-acetic acid (DNPA), which serves as a critical branch point. The configuration at C-3 of DNPA is a key determinant of structural diversity, leading to either (S)- or (R)-DNPA. Actinorhodin and medermycin are generated via (S)-DNPA, whereas granaticin is produced via (R)-DNPA. In contrast to the ketoreductase ActVI–ORF1 involved in (S)-DNPA production, the enzymatic function of Gra-6, presumed to be associated with (R)-DNPA biosynthesis, remains uncharacterized. This study evaluated the DNPA-producing ability of Gra-6 using a recombinant enzyme system expressed in Escherichia coli, revealing that it catalyzes stereoselective (R)-DNPA production. Furthermore, the putative amino acid residues involved in the DNPA-producing ability of Gra-6 were predicted through molecular modeling, including homology modeling and docking simulations with the coenzyme and an acyl carrier protein (ACP)-free bicyclic intermediate (BI) as the substrate. The involvement of these residues was verified by employing Gra-6 mutants. Additionally, the stereoselective mechanisms underlying DNPA production by each enzyme were proposed by comparing the docking models of ActVI–ORF1 and Gra-6 with the coenzyme and ACP-free BI. These findings establish Gra-6 as a pivotal stereospecific branch-point enzyme in BIQ polyketide biosynthetic branching and provide new insights into the stereoselective catalytic mechanisms.

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Acknowledgements

This work was supported by JSPS KAKENHI grants No. JP23K06200 (to K. Ishikawa) and JP 24K09883 (to K. Ichinose).

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Authors and Affiliations

  1. Faculty of Pharmaceutical Sciences, Teikyo Heisei University, Nakano-ku, Japan

    Kazuki Ishikawa

  2. Research Institute of Pharmaceutical Sciences, Musashino University, Nishitokyo-shi, Japan

    Kazuki Ishikawa, Minori Hashimoto, Noriko Kusuoku, Chiharu Nozaki, Makoto Hashimoto & Koji Ichinose

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  1. Kazuki Ishikawa
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  2. Minori Hashimoto
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  3. Noriko Kusuoku
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  4. Chiharu Nozaki
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  5. Makoto Hashimoto
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  6. Koji Ichinose
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Contributions

K. Ishikawa, Conceptualization, Data curation, Funding acquisition, Investigation, Methodology, Writing – original draft, Writing – review & editing. M. Hashimoto, N. Kusuoku and C. Nozaki, investigation. M. Hashimoto and K. Ichinose, Funding acquisition and Writing – review & editing.

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Correspondence to Kazuki Ishikawa or Koji Ichinose.

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Ishikawa, K., Hashimoto, M., Kusuoku, N. et al. Functional characterization of Gra-6 as a branch-point enzyme directing (R)-DNPA formation in BIQ polyketide biosynthesis. J Antibiot (2026). https://doi.org/10.1038/s41429-026-00912-4

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