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oai:repisalud.isciii.es:20.500.12105/97962024-09-27T21:32:01Zcom_20.500.12105_2052com_20.500.12105_2051col_20.500.12105_19609

00925njm 22002777a 4500 dc Sikorra, Stefan author Skiba, Martin author Dorner, Martin B author Weisemann, Jasmin author Weil, Mirjam author Valdezate, Sylvia author Davletov, Bazbek author Rummel, Andreas author Dorner, Brigitte G author Binz, Thomas author 2018 In the recent past, about 40 botulinum neurotoxin (BoNT) subtypes belonging to serotypes A, B, E, and F pathogenic to humans were identified among hundreds of independent isolates. BoNTs are the etiological factors of botulism and represent potential bioweapons; however, they are also recognized pharmaceuticals for the efficient counteraction of hyperactive nerve terminals in a variety of human diseases. The detailed biochemical characterization of subtypes as the basis for development of suitable countermeasures and possible novel therapeutic applications is lagging behind the increase in new subtypes. Here, we report the primary structure of a ninth subtype of BoNT/F. Its amino-acid sequence diverges by at least 8.4% at the holotoxin and 13.4% at the enzymatic domain level from all other known BoNT/F subtypes. We found that BoNT/F9 shares the scissile Q58/K59 bond in its substrate vesicle associated membrane protein 2 with the prototype BoNT/F1. Comparative biochemical analyses of four BoNT/F enzymatic domains showed that the catalytic efficiencies decrease in the order F1 > F7 > F9 > F6, and vary by up to a factor of eight. KM values increase in the order F1 > F9 > F6 ≈ F7, whereas kcat decreases in the order F7 > F1 > F9 > F6. Comparative substrate scanning mutagenesis studies revealed a unique pattern of crucial substrate residues for each subtype. Based upon structural coordinates of F1 bound to an inhibitor polypeptide, the mutational analyses suggest different substrate interactions in the substrate binding channel of each subtype. Toxins (Basel). 2018 Aug 1;10(8). pii: E311. 10.3390/toxins10080311 2072-6651 2072-6651 Toxins 30071628 http://hdl.handle.net/20.500.12105/9796 Clostridium botulinum Zn2+ protease Botulinum neurotoxin Serotype F Subtype Synaptobrevin Vesicle associated membrane protein 2 (VAMP-2) Botulinum Neurotoxin F Subtypes Cleaving the VAMP-2 Q58⁻K59 Peptide Bond Exhibit Unique Catalytic Properties and Substrate Specificities