Mechanistic insights and stucture engineering of a reductive cyclase in ikarugamycin biosynthesis

Polycyclic tetramate macrolactams (PTMs), a widely encountered family of natural products with diverse bioactivites, feature a macrocyclic lactam with an embedded tetramic acid ring. PTMs display further structural diversity by fusing the macrolactam ring with a different set of carbocyclic rings. Although a number of PTM biosynthetic gene clusters have been characterized or implicated in diverse bacteria, the biochemical mechanisms for the construction of polycyclic ring systems in PTMs remain enigmatic. Ikarugamycin is a PTM family member and has attracted extensive attentions as a promising lead in anticancer therapeutics since its discovery in early 1970s. Recently, we have reisolated ikarugamycin from a marine Streptomyces sp. ZJ306 derived from the Pearl River estuary. Isotope labeling studies confirmed that ikarugamycin biosynthesis was originated from an unusual hybrid polyketide synthase/nonribosomal peptide synthetase (PKS/NRPS), consistent with other PTMs. Subsequently, dissection and reconstitution of ikarugamycin biosynthesis by in vivo gene disruption and heterologous expression studies not only identified a minimal three-gene cassette ikaABC capable of conferring the heterologous production of ikarugamycin, but also established the function and reaction timing of IkaABC. Next, the biochemical mechanism of the construction of an inner five-membered ring in ikarugamycin, catalyzed by the NAD(P)H-dehydrogenase IkaC, was unveiled by labelling studies using stereospecifically deuterated NADPH cofactor and/or deuterium oxide, which led to a mechanistic proposal involving an unusual [1 + 6] Michael addition reaction. Finally, crystal structure-based protein engineering of the cyclase IkaC afforded several variants that are able to produce new ikarugamycin analogues.