Site-directed mutagenesis identifies active-site residues of the light chain of botulinum neurotoxin type A

Botulinum neurotoxins (BoNTs) are metalloproteases which block neuroexocyto sis via specific cleavage and inactivation of SNARE proteins. Such proteoly sis accounts for the extreme toxicity of these neurotoxins and of their pro longed effect. The recently determined structures of BoNT/A and/B allows on e to design active-site mutants to probe the role of specific residues in t he proteolysis of SNARE proteins. Here we present the results of mutations of the second glutamyl residue involved in zinc coordination and of a tyros ine and a phenylalanine residues that occupy critical positions within the active site of BoNT/A. The spectroscopic properties of the purified mutants are closely similar to those of the wild-type molecule indicating the acqu isition of a correct tertiary structure. Mutation of the Glu-262* nearly ab olishes SNAP-25 hydrolysis as expected for a residue involved in zinc coord ination. The Phe-266 and Tyr-366 mutants have reduced proteolytic activity indicating a direct participation in the proteolytic reaction, and their po ssible role in catalysis is discussed. (C) 2001 Academic Press.