片山 欣哉

Mutations in the Streptomyces peucetius dnrD gene block the ring cyclization leading from aklanonic acid methyl ester to aklaviketone, an intermediate in the biosynthetic pathway to daunorubicin and doxorubicin. To investigate the role of DnrD in this transformation its gene was overexpressed in Escherichia coli and the DnrD protein was purified to homogeneity and characterized. The enzyme was shown to catalyse the conversion of aklanonic acid methyl ester to aklaviketone presumably via an intramolecular aldol condensation mechanism. In contrast to the analogous intramolecular aldol cyclization catalyzed by the TcmI protein from the tetracenomycin C pathway in Streptomyces glaucescens, where a tricyclic anthraquinol carboxylic acid is converted to its fully aromatic tetracyclic form, the conversion catalyzed by DnrD occurs after anthraquinone formation and requires activation of a carboxylic acid group by esterification of aklanonic acid, the aklanonic acid methyl ester precursor. Also, the cyclization is not coupled with a subsequent dehydration step that would result in an aromatic ring. As the substrates for the DnrD and TcmI enzymes are among the earliest isolable intermediates of aromatic polyketide biosynthesis, an understanding of the mechanism and active site topology of these proteins will allow one to determine the substrate and mechanistic parameters important for aromatic ring formation. In the future, these parameters may be able to be applied to some of the earlier polyketide cyclization processes that currently are difficult to study in vitro.

First enzyme which catalyzes Diels-Alder reaction has been partially purified. This crude enzyme is able to catalyze [4+2]-cycloaddition of prosolanapyrone III to the exo adduct solanapyrone A and the endo adduct solanapyrone D in a 7:1 ratio differing substantially from the ratio found in a background reaction (exo/endo, 1:23). The optical purity of the enzymatic reaction product solanapyrone A was estimated as 92%ee by HPLC analysis monitored with CD spectrometer. The enzyme also exhibited an oxidase activity of prosolanapyrone II to III. Two-step conversion of prosolanapyrone II to solanapyrones A and B by the crude enzyme gave better diastereoselectivity and enantioselectivity (A: 99%ee). A high reaction rate of the background reaction for prosolanapyrone III caused some losses in both diastereo- and enantioselectivity. Thus, we suggest that a specific enzyme catalyzes both oxidation and Diels-Alder reaction producing enantiomerically pure solanapyrones.