Identification of the biosynthetic gene cluster for the antiviral spirotetronate MM46115, and investigation of a putative diene forming dehydratase domain Rebecca Clayton 1 , Matthew Jenner 1 , Lona M. Alkhalaf 1 and Gregory L. Challis 1,2 1 University of Warwick, UK, 2 Monash University, Australia Natural products are assembled by highly selective and efficient biosynthetic enzymes, which carry out chemical transformations that are often difficult to accomplish synthetically. Understanding the mechanisms of biosynthetic enzymes improves their potential to be exploited for the sustainable production of novel complex molecules, such as those with pharmaceutical or agrochemical value. Spirotetronates are a class of natural products containing spiro-fused cyclohexene and tetronic acid moieties. MM46115 is one such example that is reported to have activity against parainfluenza viruses 1 and 2, and Gram-positive bacteria 1 .Here we report identification of the MM46115 biosynthetic gene cluster in Actinomadura pelletieri DSM 43383. Analysis of the gene cluster identified genes that are predicted to encode biosynthetic enzymes involved in production of the polyketide backbone, the deoxy sugar, and a chlorinated 6-methyl salicylic acid derivative. Additionally, two enzymes proposed to catalyse intramolecular Diels-Alder reactions have been identified. Bioinformatic analysis of the acetyltransferase (AT), dehydratase (DH), ketoreductase (KR) and enoylreductase (ER) domains of the polyketide synthase (PKS), was used to predict the structure of the expected product of this modular multienzyme. This corresponds closely to the putative polyketide precursor of MM46115, 2 except for an alkene, which is missing from the predicted product of the PKS, but is required to form the spirotetronate. We propose that the DH domain in the downstream module catalyses double dehydration of a 3, 5-dihydroxy thioester to install the requisite diene. Here we present our efforts to investigate the catalytic activity of this DH domain in vitro , using a combination of purified recombinant proteins, chemically synthesised substrates and intact protein mass spectrometry. References 1. R. J. Ashton, M. D. Kenig, K. Luk, D. N. Planterose and G. Scott-Wood, J. Antibiot. (Tokyo). , 1990, 43 , 1387–1393. 2. K. Luk and S. A. Readshaw, J. Chem. Soc., Perkin Trans , 1991, 1 , 1641–1644.
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