Analysis of active site residues of botulinum neurotoxin E by mutational, functional, and structural studies: Glu335Gln is an apoenzyme.
Clostridial neurotoxins comprising the seven serotypes of botulinum neurotoxins and tetanus neurotoxin are the most potent toxins known to humans. Their potency coupled with their specificity and selectivity underscores the importance in understanding their mechanism of action in order to develop a strategy for designing counter measures against them. To develop an effective vaccine against the toxin, it is imperative to achieve an inactive form of the protein which preserves the overall conformation and immunogenicity. Inactive mutants can be achieved either by targeting active site residues or by modifying the surface charges farther away from the active site. The latter affects the long-range forces such as electrostatic potentials in a subtle way without disturbing the structural integrity of the toxin causing some drastic changes in the activity/environment. Here we report structural and biochemical analysis on several mutations on Clostridium botulinum neurotoxin type E light chain with at least two producing dramatic effects: Glu335Gln causes the toxin to transform into a persistent apoenzyme devoid of zinc, and Tyr350Ala has no hydrolytic activity. The structural analysis of several mutants has led to a better understanding of the catalytic mechanism of this family of proteins. The residues forming the S1' subsite have been identified by comparing this structure with a thermolysin-inhibitor complex structure.
Agarwal
Rakhi
R
Biology Department, Brookhaven National Laboratory, Upton, New York 11973, USA.
Binz
Thomas
T
Swaminathan
Subramanyam
S
D000409:Alanine / Q000235:genetics; D001051:Apoenzymes / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D001120:Arginine / Q000235:genetics; D001216:Asparagine / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D001905:Botulinum Toxins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D002384:Catalysis; D003460:Crystallization; D004789:Enzyme Activation / Q000235:genetics; D018698:Glutamic Acid / Q000235:genetics; D005973:Glutamine / Q000235:genetics; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D015260:Neprilysin / Q000737:chemistry / Q000378:metabolism; D009498:Neurotoxins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011480:Protease Inhibitors / Q000737:chemistry; D013379:Substrate Specificity / Q000235:genetics; D013820:Thermolysin / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D013912:Threonine / Q000235:genetics; D014443:Tyrosine / Q000235:genetics
D001051:Apoenzymes; D009498:Neurotoxins; D011480:Protease Inhibitors; D005973:Glutamine; D013912:Threonine; D018698:Glutamic Acid; D014443:Tyrosine; D001216:Asparagine; D001120:Arginine; D015260:Neprilysin; D013820:Thermolysin; D001905:Botulinum Toxins; D000409:Alanine; C096326:botulinum toxin type E