Abstract
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.
Highlights
Botulinum neurotoxins (BoNTs) are produced by the Gram-positive bacterium, Clostridium botulinum, and some other strains of C. baratii and C. butyricum
Comparison of the amino-acid sequence with other botulinum neurotoxin (BoNT) sequences available in GenBank revealed that its sequence is related to BoNT/F and exhibits between 91.6% and 69.0% identity to the published BoNT/F subtypes F1–F8 (Table 1) [7,8]
The amino-acid sequence identities among BoNT/F9 and all known subtypes, including the mosaic BoNT/H
Summary
Botulinum neurotoxins (BoNTs) are produced by the Gram-positive bacterium, Clostridium botulinum, and some other strains of C. baratii and C. butyricum. They are differentiated based on serum neutralization properties into seven accepted serotypes (A to G). A still rapidly growing number of BoNT subtypes were identified in the last decade based on sequence diversity, and were assigned to serotypes A, B, E, and F [4,5]. BoNT sequences with at least 2.6% difference in amino-acid sequence were designated as individual subtypes [6]. BoNT/F represents the serotype with the largest sequence variations among its subtypes. The eight characterized subtypes (BoNT/F1–F8; [7,8]) to date exhibit as far as 36% divergence
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