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

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.