Active-site mutagenesis of tetanus neurotoxin implicates TYR-375 and GLU-271 in metalloproteolytic activity

摘要: Tetanus neurotoxin (TeNT) blocks neurotransmitter release by cleaving VAMP/synaptobrevin, a membrane associated protein involved in synaptic vesicle fusion. Such activity is exerted the N-terminal 50kDa domain of TeNT which zinc-dependent endopeptidase (TeNT-L-chain). Based on three-dimensional structure botulinum serotype A (BoNT/A) and B (BoNT/B), two proteins closely related to TeNT, X-ray scattering studies we have designed mutations at active site residues probe their involvement activity. The metalloproteases composed primary sphere co-ordinating zinc atom, secondary that determines proteolytic specificity Glu-261 Glu-267 directly co-ordinates atom BoNT/A BoNT/B respectively corresponding residue was replaced Asp or non conservative Ala. Tyr-365 4.3A away from BoNT/A, Phe purified mutants had CD, fluorescence UV spectra similar those wild-type molecule. TeNT-Asp-271 (E271D) native molecule, whereas TeNT-Phe-375 (Y375F) lower than control. Interestingly, Ala are completely devoid enzymatic These results demonstrate both Glu-271 Tyr-375 essential for TeNT.