Makoto Hashimoto

Actinorhodin (ACT) from Streptomyces coelicolor A3(2) is an aromatic polyketide antibiotic with a benzoisochromanequinone (BIQ) skeleton. Although actVI-ORF3 and actVI-ORF4 are not essential for ACT biosynthesis, homologous genes to these are present in the biosynthetic gene clusters of BIQ lactones. In this study, ActVI-ORF3 was identified as a cofactor-independent enzyme with lactonization activity, using ACT as a substrate. ActVI-ORF3 recognized dihydrokalafungin and 8-hydroxykalafafungin, which share the same pyran-ring configuration as ACT, but not nanaomycin A, which has an opposite configuration. In contrast, ActVI-ORF4 functioned as an NAD(P)-dependent oxidoreductase, catalyzing the delactonization of BIQ lactones. Conversion experiments using isotopically labeled compounds revealed that both lactonization and delactonization reactions of these enzymes yielded products in which the carboxyl oxygen at the C1 position was retained. Subsequently, we reexamined the accumulation of ACT-related compounds in the actVI-ORF3 and actVI-ORF-4 disruptants. The results suggested that ACT intermediates are predominantly pooled in the bacteria as (S)-DNPA rather than in lactone-form. The contribution of ActVI-ORF4 to metabolic flux is not significant, and endogenous reductases can convert these intermediates to the dihydro form, which subsequently re-enters the ACT biosynthetic pathway.

A plethora of dimeric natural products exist with diverse chemical structures and biological activities. A major strategy for dimerization is aryl coupling reactions catalyzed by cytochrome P450 or laccase. Actinorhodin (ACT) from Streptomyces coelicolor has a dimeric pyranonaphthoquinone structure connected by a C-C bond. Here, we identified a NmrA-family dimerizing enzyme, ActVA-ORF4, and a cofactor independent oxidase, ActVA-ORF3, both involved in the last step of ACT biosynthesis. ActVA-ORF4 is a unique  NAD(P)H-dependent enzyme that catalyzes the inter-molecular C-C bond formation using 8-hydroxydihydrokalafungin (DHK-OH) as the sole substrate. On the other hand, ActVA-ORF3 was found to be a quinone-forming enzyme that produces the coupling substrate, DHK-OH, and the final product, ACT. Consequently, the functional assignment of all essential enzymes in ACT biosynthesis was completed, which would be a landmark in our understanding of the entire biosynthetic pathway for one of the best-known model natural products, ACT.