Development of a second-generation 13C-NMR assay to study β-branch formation in the kalimantacin antibiotics Angus Weir 1 , Joleen Masschelein 1 , Matthew P. Crump 2 , Chris L. Willis 2 1 KU Leuven - VIB, Belgium, 2 University of Bristol,UK The kalimantacin antibiotics exhibit strong selective activity against staphylococcus , including resistant strains. 1 Of particular interest are unique aspects of kalimantacin biosynthesis, which includes the selective installation of three consecutive β-branches, features that structurally diversify polyketides. Their installation involves the interplay of the regular polyketide assembly components with a multi-enzyme trans -acting cassette. 2 Previously we have detailed the mechanism and complex protein-protein interactions required for selective branch installation, through a first-generation real-time 1,3 C NMR assay coupled with mass spectrometry. 3 This assay has now been complemented with the development of a second-generation 13 C NMR assay to further study the mechanism behind selective branch formation, specifically the decarboxylation and protonation controlled by Enoyl-CoA-Hydratase (ECH) domains. The development and results generated from this second-generation assay along with in vivo mutant studies will be presented. References 1. Fage, C. D.; Lathouwers, T.; Vanmeert, M.; Gao, L-J.; Vrancken, K.; Lammens, E-M., Weir, A. N. M., Degroote, R.; Cuppens, H.; Kosol, S.; Simpson, T. J.; Crump, M. P.; Willis, C. L.; Herdewijn, P.; Lescrinier, E.; Lavigne, R.; Anné, J.; Masschelein, J.; Angew. Chem. Int. Ed., 2020, 59 , 26, 10549. 2. Walker, P. D.; Weir, A. N. M.; Willis, C. L.; Crump, M. P.; Nat. Prod. Rep. , 2021, 38 , 723. 3. Walker, P.D.; Williams, C.; Weir, A. N. M.; Wang, L.; Crosby, J.; Race, P. R.; Simpson, T. J.; Willis, C. L.; Crump, M. P.; Angew. Chem. Int. Ed. , 2019, 58 , 36, 12446.
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