Evidence that a Novel Thioesterase is Responsible for Polyketide Chain Release during Biosynthesis of the Polyether Ionophore Monensin

Abstract

Polyether ionophores, such as monensin A, are known to be biosynthesised, like many other antibiotic polyketides, on giant modular polyketide synthases (PKSs), but the intermediates and enzymes involved in the subsequent steps of oxidative cyclisation remain undefined. In particular there has been no agreement on the mechanism and timing of the final polyketide chain release. We now report evidence that MonCII from the monensin biosynthetic gene cluster in Streptomyces cinnamonensis, which was previously thought to be an epoxide hydrolase, is a novel thioesterase that belongs to the alpha/beta-hydrolase structural family and might catalyse this step. Purified recombinant MonCII was found to hydrolyse several thioester substrates, including an N-acetylcysteamine thioester derivative of monensin A. Further, incubation with a hallmark inhibitor of such enzymes, phenylmethanesulfonyl fluoride, led to inhibition of the thioesterase activity and to the accumulation of an acylated form of MonCII. These findings require a reassessment of the role of other enzymes implicated in the late stages of polyether ionophore biosynthesis.

Cloning and Characterization of the Polyether Salinomycin Biosynthesis Gene Cluster of Streptomyces albus XM211

“…Due to the absence of a conventional TE domain within the PKS or a NanE-like thioesterase (22,23), there are three candidates for the requisite thioesterase activity in the salinomycin biosynthesis gene cluster. Genes slnDI and slnDII, encoding discrete type II TEs, display high similarity to monAIX and monAX in the monensin gene cluster (14). The slnDI disruption mutant JCY23 produced salinomycin at a level similar to that of wild-type XM211 (Fig.…”

Identification of a Gene Cluster of Polyether Antibiotic Lasalocid fromStreptomyces lasaliensis

“…Recently, several experimental results using discrete TE have strongly indicated that cleavage of polyketide-tethered ACP occurred after polyether formation. [49][50][51] The finding of a TE domain at the Cterminal end of Lsd17 (Fig. 2) is noteworthy, since this suggests that the full-length polyketide is cleaved from the ACP domain, as is usual for polyketide metabolites, before polyether formation.…”

“…One major difficulty in elucidating details of the reaction mechanism for polyether formation in the biosynthesis of monensin-type ionophore antibiotics is the involvement of complex modification reactions such as full-length polyketide transfer to discrete ACP, 17) oxidative cascade cyclization of the polyketide bound to ACP, subsequent hydrolysis of discrete TE, [49][50][51] methylation and hydroxylation. In this report, we have identified the gene cluster of another representative ionophore antibiotic, lasalocid.…”

Die biosynthetische Grundlage der Polyketid‐Vielfalt

“…[211] Auf Grundlage von TE-Proteinstrukturen schlugen Boddy et al vor, dass hydrophobe Wechselwirkungen zwischen dem Bindungshohlraum und dem Substrat die Substratspezifität lenken und damit die Größe des Makrolactons bestimmen. [214] In den Biosyntheseapparaten der Polyether Nanchangmycin [215,216] und Monensin [217] wurden ungewöhnliche Thioesterasen, die zu linearen Produkten führen, identifiziert. Für die Synthese des makrocyclischen Antibiotikums Lankacidin (89) in Streptomyces rochei wurde ein alternativer Cyclisierungsmechanismus vorgeschlagen (Schema 39).…”