DnrD cyclase involved in the biosynthesis of doxorubicin: Purification andcharacterization of the recombinant enzyme

Mutations in the Streptomyces peucetius dnrD gene block the ring cyclizatio n leading from aklanonic acid methyl ester (AAME) to aklaviketone (AK), an intermediate in the biosynthetic pathway to daunorubicin (DNR) and doxorubi cin. To investigate the role of DnrD in this transformation, its gene was o verexpressed in Escherichia coli and the DnrD protein was purified to homog eneity and characterized. The enzyme was shown to catalyze the conversion o f AAME to AK presumably via an intramolecular aldol condensation mechanism. In contrast to the analogous intramolecular aldol cyclization catalyzed by the TcmI protein from the tetracenomycin (TCM) C pathway in Streptomyces g laucescens, where a tricyclic anthraquinol carboxylic acid is converted to its fully aromatic tetracyclic form, the conversion catalyzed by DnrD occur s after anthraquinone formation and requires activation of a carboxylic aci d group by esterification of aklanonic acid, the AAME precursor. Also, the cyclization is not coupled with a subsequent dehydration step that would re sult in an aromatic ring. As the substrates for the DnrD and TcmI enzymes a re among the earliest isolable intermediates of aromatic polyketide biosynt hesis, an understanding of the mechanism and active site topology of these proteins will allow one to determine the substrate and mechanistic paramete rs that are important for aromatic ring formation. In the future, these par ameters may be able to be applied to some of the earlier polyketide cycliza tion processes that currently are difficult to study in vitro.