Lethal factor active-site mutations affect catalytic activity in vitro

Area of Science:

• Biochemistry
• Microbiology
• Proteomics

Background:

• Bacillus anthracis lethal toxin's lethal factor (LF) possesses a zinc-binding motif (HEXXH) and is hypothesized to be a Zn2+ metalloprotease.
• Previous research suggested LF's function in macrophages, but direct proteolytic activity on substrates remained unproven.

Purpose of the Study:

• To investigate the in vitro proteolytic activity of Bacillus anthracis lethal factor (LF).
• To identify potential substrates and characterize the catalytic mechanism of LF.

Main Methods:

• In vitro hydrolysis of synthetic peptides by purified LF.
• Analysis of cleavage products using mass spectrometry and high-pressure liquid chromatography (HPLC).
• Site-directed mutagenesis of active-site residues and inhibition assays with various chelators and protease inhibitors.

Main Results:

• LF demonstrated proteolytic activity, hydrolyzing synthetic peptides, with a preference for proline-containing substrates.
• Mutations in active-site residues and the addition of zinc chelators (1,10-phenanthroline, EDTA) abolished LF activity.
• Specific inhibitors like ZINCOV blocked LF activity, while others (bestatin, lysine CMK) did not affect in vitro function.

Conclusions:

• These findings provide the first direct evidence that Bacillus anthracis LF functions as an endopeptidase.
• LF's catalytic activity is dependent on Zn2+ and its active-site residues.
• The substrate preference for proline-containing peptides offers insights into LF's mechanism of action.