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Infect Immun, May 1998, p. 2374-2378, Vol. 66, No. 5
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Lethal Factor Active-Site Mutations Affect Catalytic Activity In Vitro

S. E. Hammond1 and P. C. Hanna1,2,*

Department of Microbiology1 and Department of Immunology,2 Duke University Medical Center, Durham, North Carolina 27710

Received 26 June 1997/Returned for modification 11 August 1997/Accepted 31 January 1998

The lethal factor (LF) protein of Bacillus anthracis lethal toxin contains the thermolysin-like active-site and zinc-binding consensus motif HEXXH (K. R. Klimpel, N. Arora, and S. H. Leppla, Mol. Microbiol. 13:1093-1100, 1994). LF is hypothesized to act as a Zn2+ metalloprotease in the cytoplasm of macrophages, but no proteolytic activities have been previously shown on any target substrate. Here, synthetic peptides are hydrolyzed by LF in vitro. Mass spectroscopy and peptide sequencing of isolated cleavage products separated by reverse-phase high-pressure liquid chromatography indicate that LF seems to prefer proline-containing substrates. Substitution mutations within the consensus active-site residues completely abolish all in vitro catalytic functions, as does addition of 1,10-phenanthroline, EDTA, and certain amino acid hydroxamates, including the novel zinc metalloprotease inhibitor ZINCOV. In contrast, the protease inhibitors bestatin and lysine CMK, previously shown to block LF activity on macrophages, did not block LF activity in vitro. These data provide the first direct evidence that LF may act as an endopeptidase.

* Corresponding author. Mailing address: Department of Microbiology, Duke University Medical Center, Box 3020, Durham, NC 27710. Phone: (919) 681-6702. Fax: (919) 684-8735. E-mail: hanna{at}abacus.mc.duke.edu.

Infect Immun, May 1998, p. 2374-2378, Vol. 66, No. 5
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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