Preparation and characterization of cobalt-substituted anthrax lethal factor

• Published in: Biochemical and biophysical research communications Vol. 416; no. 1; pp. 106 - 110
• Main Authors: Säbel, Crystal E., Carbone, Ryan, Dabous, John R., Lo, Suet Y., Siemann, Stefan
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 09.12.2011
• Subjects: 60 APPLIED LIFE SCIENCES; Anthrax lethal factor; Antigens, Bacterial - chemistry; BACILLUS MEGATERIUM; Bacillus megaterium - enzymology; Bacillus megaterium - genetics; Bacillus megaterium - growth & development; Bacterial Toxins - chemistry; Catalytic Domain; Cell Culture Techniques; COBALT; Cobalt - chemistry; Cobalt - metabolism; COBALT CHLORIDES; COBALT IONS; CRYSTAL STRUCTURE; Electronic spectroscopy; INORGANIC, ORGANIC, PHYSICAL AND ANALYTICAL CHEMISTRY; Kinetics; Metal substitution; Metalloendopeptidases - chemistry; PHOSPHOTRANSFERASES; SPECTRA; Spectrophotometry; SPECTROSCOPY; THERMAL GRAVIMETRIC ANALYSIS; Thioglycolic acid; ZINC; Zinc - chemistry; Zinc proteases; Electronic spectroscopy; Zinc proteases; Anthrax lethal factor; Metal substitution; Thioglycolic acid
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2011.11.005

► Cobalt-substituted anthrax lethal factor (CoLF) is highly active. ► CoLF can be prepared by bio-assimilation and direct exchange. ► Lethal factor binds cobalt tightly. ► The electronic spectrum of CoLF reveals penta-coordination. ► Interaction of CoLF with thioglycolic acid follows a 2-step mechanism. Anthrax lethal factor (LF) is a zinc-dependent endopeptidase involved in the cleavage of mitogen-activated protein kinase kinases near their N-termini. The current report concerns the preparation of cobalt-substituted LF (CoLF) and its characterization by electronic spectroscopy. Two strategies to produce CoLF were explored, including (i) a bio-assimilation approach involving the cultivation of LF-expressing Bacillus megaterium cells in the presence of CoCl 2, and (ii) direct exchange by treatment of zinc-LF with CoCl 2. Independent of the method employed, the protein was found to contain one Co 2+ per LF molecule, and was shown to be twice as active as its native zinc counterpart. The electronic spectrum of CoLF suggests the Co 2+ ion to be five-coordinate, an observation similar to that reported for other Co 2+-substituted gluzincins, but distinct from that documented for the crystal structure of native LF. Furthermore, spectroscopic studies following the exposure of CoLF to thioglycolic acid (TGA) revealed a sequential mechanism of metal removal from LF, which likely involves the formation of an enzyme: Co 2+:TGA ternary complex prior to demetallation of the active site. CoLF reported herein constitutes the first spectroscopic probe of LF’s active site, which may be utilized in future studies to gain further insight into the enzyme’s mechanism and inhibitor interactions.