Carbon-carbon double-bond shift in the biosynthesis of the antibiotic corallopyronin A

Abstract

Corallopyronin A is a myxobacterial compound, which was isolated in our lab from the strain Corallococcus coralloides B035. It is a potent in vivo active antibiotic, which is currently undergoing preclinical studies. Regarding its biosynthesis, corallopyronin A was found to originate from two chains, one being solely PKS- and the other NRPS/PKS dervived.<br /> In polyketide biosynthesis the reduction of β-carbonyl groups to an alkene moiety usually results in a α,β positioned double-bond. However, in a few polyketides the rare case of such a carbon-carbon double-bond in β,γ position is depicted, e.g. in the biosynthesis of ansamitocin (Taft et al., 2009), bacillaene (Moldenhauer et al., 2010) and rhizoxin (Kusebauch et al., 2010). For rhizoxin it was shown that the respective double-bond (Δ8) was shifted to the β,γ position after elongation of the nascent polyketide chain by a distinct “shift module” including an unusual dehydratase-like domain (DH*) downstream in the PKS assembly line (Kusebauch et al., 2010). We proposed a similar process for the antibiotic corallopyronin A and provided here evidence that a distinct domain (CorJ DH*) catalyses the carbon-carbon double-bond shift from α,β to β,γ position during corallopyronin A biosynthesis.<br /> In this study the in vitro analysis of the enzyme domain (CorJ DH*) responsible for this double-bond isomerisation was analysed. This “shift domain” was heterologously expressed and assayed with its acyl carrier protein bound substrate. To facilitate this analysis the biosynthetic corallopyronin A intermediate was chemically synthesized as a N-acetylcysteamine-thioester.<br /> Enzyme activity was analyzed by NMR and high-resolution MS measurements, the latter were possible by performing the assay in deuterated buffer, thereby observing a proton/deuterium exchange reaction. The here reported in vitro experiments clearly demonstrated that CorJ DH* acts as double-bond migrating enzyme in corallopyronin A biosynthesis. Mutated enzyme variants (CorJ DH*H47A and CorJ DH*D211N) gave first experimental evidence for the essential amino acids involved in double-bond migration. It could be shown that the amino acid histidine in position 47 (H47) plays a major role in the double-bond isomerisation in that it serves as proton donor. A still unknown residue must function as acceptor, which is in agreement with the mechanism postulated earlier by Hertweck and co-workers (Kusebauch et al., 2010).<br /> These results provide evidence for the genetic and enzymatic basis of carbon-carbon double-bond migrations in polyketides. Furthermore, they support the partly still hypothetical corallopyronin A biosynthetic process, and widen the understanding of PKS systems in general as the tool box for the rational design of metabolites in genetic engineering (Lohr et al., 2013).