title: Engineering Clostridia Neurotoxins with elevated catalytic activity.
abstract: BoNT/B and TeNT cleave substrate VAMP2 at the same scissile bond, yet these two toxins showed different efficiency on substrate hydrolysis and had different requirements for the recognition of P2' site of VAMP2, E(78). These differences may be due to their different composition of their substrate recognition pockets in the active site. Swapping of LC/T S1' pocket residue, L(230), with the corresponding isoleucine in LC/B increased LC/T activity by ∼25 fold, while swapping of LC/B S1' pocket residue, S(201), with the corresponding proline in LC/T increased LC/B activity by ∼10 fold. Optimization of both S1 and S1' pocket residues of LC/T, LC/T (K(168)E, L(230)I) elevated LC/T activity by more than 100-fold. The highly active LC/T derivative engineered in this study has the potential to be used as a more effective tool to study mechanisms of exocytosis in central neuron. The LC/B derivative with elevated activity has the potential to be developed into novel therapy to minimize the impact of immunoresistance during BoNT/B therapy.
authors.affiliation: Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong Special Administrative Region.
authors.forename: Jiubiao
authors.initials: J
authors.lastname: Guo
authors.forename: Xuehua
authors.initials: X
authors.lastname: Pan
authors.forename: Yanxiang
authors.initials: Y
authors.lastname: Zhao
authors.forename: Sheng
authors.initials: S
authors.lastname: Chen
mesh_terms: D000818:Animals; D002384:Catalysis; D020134:Catalytic Domain; D045744:Cell Line, Tumor; D003460:Crystallization; D006868:Hydrolysis; D051379:Mice; D009498:Neurotoxins / Q000737:chemistry* / Q000235:genetics; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013744:Tetanus Toxin / Q000737:chemistry* / Q000235:genetics; D050988:Vesicle-Associated Membrane Protein 2 / Q000235:genetics / Q000378:metabolism
chemical_list: D009498:Neurotoxins; D011994:Recombinant Proteins; D013744:Tetanus Toxin; D050988:Vesicle-Associated Membrane Protein 2; C494808:vesicle-associated membrane protein 2, mouse
keywords: BoNT/B; Botulinum Neurotoxin; Botulinum Neurotoxin B; CNTs; Clostridia Neurotoxins; Elevated activity; Engineering; LC; Light chain; SNARE; TeNT; Tetanus Neurotoxin; VAMP2; light chain; soluble NSF attachment receptor; vesicle associated membrane protein-2