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Infection and Immunity, November 2000, p. 6378-6383, Vol. 68, No. 11
Institut für Experimentelle und
Klinische Pharmakologie und Toxikologie,
Albert-Ludwigs-Universität Freiburg, D-79104 Freiburg,
Germany
Received 1 May 2000/Returned for modification 16 June 2000/Accepted 4 August 2000
Clostridium novyi alpha-toxin belongs to the family of
large clostridial cytotoxins which act on cells through the
modification of small GTP-binding proteins. We present here an analysis
of the catalytic domain of alpha-toxin. A NH2-terminal
551-amino-acid fragment,
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Characterization of the Catalytic Domain of
Clostridium novyi Alpha-Toxin
551, was found to contain the full enzyme
activity of the holotoxin, whereas a slightly shortened fragment
encompassing 509 amino acids showed no detectable enzyme activity.
Further characterization of the enzymatically active fragment 551
revealed a substrate specificity for both
UDP-N-acetylglucosamine and UDP-glucose. A
Michaelis-Menten constant of 17 µM was determined for the substrate UDP-N-acetylglucosamine, while that for UDP-glucose was
about 20 times higher, indicating a weaker affinity of the toxin for the latter substrate. Mutation of the aspartic acids of a conserved motif DXD within 551 reduced enzyme activity >700-fold and
inhibited cytotoxicity after microinjection in cells. Inhibition of
enzyme activity of the DXD mutant could be partially overcome by
increased concentrations of manganese ions, suggesting the involvement
of these aspartic acids in Mn2+ binding. By construction of
chimeras of enzymatically active fragments of C. sordellii
lethal toxin and C. novyi alpha-toxin, we located the
region involved in nucleotide-sugar specificity to amino acids 133 through 517.
*
Corresponding author. Mailing address: Institut
für Experimentelle und Klinische Pharmakologie und Toxikologie,
Albert-Ludwigs-Universität Freiburg, Hermann-Herder-Str. 5, D-79104 Freiburg, Germany. Phone: 0761-2035301. Fax: 0761-2035311. E-mail: aktories{at}ruf.uni-freiburg.de.
Infection and Immunity, November 2000, p. 6378-6383, Vol. 68, No. 11
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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