Investigation of the Diels–Alderase in abyssomicin C biosynthesis Jawaher Alnawah, Christine L. Willis, Catherine Back, Shangyuwen Yang, Paul R. Race University of Bristol, UK The Diels–Alder reaction is widely used in organic chemistry to construct molecules containing a cyclohexene core. However, it often requires the use of harsh conditions such as elevated temperature and pressure or the use of catalysts. 1 An attractive alternative would be to use enzymes to catalyse such [4+2] cycloadditions at room temperature. Hence, significant effort has been dedicated to the development of artificial Diels–Alderases 2 as well as to the study of biosynthetic pathways proposed to involve a Diels-Alder reaction. 3–7 However, the field is in its infancy and significant effort is required to develop such biocatalytic carbon-carbon bond forming reactions for use on a synthetically valuable scale. The antibiotic abyssomicin C is a notable spirotetronate formed via a Diels-Alder reaction catalysed by AbyU ( Scheme1 ). 8 Using a combination of organic synthesis, X-ray crystallography and molecular modelling, we have provided insights into the mechanism of AbyU, which involves the intramolecular cycloaddition between a conjugated diene and the exo -methylene group in tetronate 1 . We have prepared an analogue 2 and shown that it is also a substrate for AbyU giving 4 Scheme 1: Diels—Alder substrate 2 converted to cyclised product 4 by AbyU. Bound molecule and key residues of AbyU binding site are highlighted. 8 This work is focused on identifying the key residues that are involved in substrate-folding within the AbyU active site. The synthesis of “non-turnover” substrates, which lacks the full triene system of 2 , will be reported alongside the crystal structure of AbyU with the bound substrate. References 1. C. Nicolaou, S. A. Snyder, T. Montagnon and G. Vassilikogiannakis, Angew. Chemie - Int. Ed. , 2002 , 41, 1668–1698. 2. Siegel, J. B.; Zanghellini, A.; Lovick, H. M.; Kiss, G.; Lambert, A. R.; St.Clair, J. L.; Gallaher, J. L.; Hilvert, D.; Gelb, M. H.; Stoddard, B. L.; Houk, K. N.; Michael, F. E.; Baker, Science , 2010 , 329, 305–309. 3. Ose, K. Watanabe, T. Mie, M. Honma, H. Watanabe, M. Yao, H. Oikawa and I. Tanaka, Nature , 2003 , 422, 185–189. 4. Auclair, A. Sutherland, J. Kennedy, D. J. Witter, J. P. Van den Heever, C. Richard Hutchinson and J. C. Vederas, J. Am. Chem. Soc. , 2000 , 122, 11519–11520. 5. R. Kim, B. Illarionov, M. Joshi, M. Cushman, | Chan, Y. Lee, W. Eisenreich, M. Fischer and A. Bacher, J. Am. Chem. Soc. , 2010 , 132, 2983–2990. J. Kim, M. W. Ruszczycky, S. H. Choi, Y. N. Liu and H. W. Liu, Nature , 2011 , 473, 109–112. 6. Hashimoto, J. Hashimoto, K. Teruya, T. Hirano, K. Shin-Ya, H. Ikeda, H.-W. Liu, M. Nishiyama and T. Kuzuyama, J. Am. Chem. Soc. , 2015 , 137, 572–575. 7. J. Byrne, N. R. Lees, L. C. Han, M. W. Van Der Kamp, A. J. Mulholland, J. E. M. Stach, C. L. Willis and P. R. Race, J. Am. Chem. Soc. , 2016 , 138, 6095–6098.
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