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MANT nucleotides as probes for analysis of Edema Factor, a bacterial Adenylyl Cyclase toxin
Author(s) -
Suryanarayana Srividya,
Tang WeiJen,
Lushington Gerald,
Richter Mark,
Seifert Roland
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.6.a807-d
Subject(s) - anthrax toxin , bacillus anthracis , adenylyl cyclase , nucleotide , enzyme , chemistry , toxin , biochemistry , microbiology and biotechnology , biology , bacteria , fusion protein , recombinant dna , gene , genetics
Anthrax is caused by Bacillus anthracis, a spore‐forming gram‐positive bacterium. Bacillus anthracis releases three exotoxins at the cell surface when it comes in contact with host cells. These toxins are edema factor (EF), lethal factor (LF) and protective antigen (PA). A synergistic action of all the three toxins causes anthrax. PA is a transport protein that mediates the entry of EF and LF into host cells. EF is a calmodulin (CaM)‐activated adenylyl cyclase that is structurally distinct from mammalian adenylyl cyclases. EF contributes significantly to both cutaneous and systemic anthrax. The crystal structure of EF bound to CaM revealed the mechanism of enzyme activation (Shen et al., EMBO J. 24 , –941, 2005). We have established a sensitive fluorescence‐based assay using several 2′, 3′‐O‐(N‐methylanthraniloyl) (MANT)‐nucleotides as probes to analyze the interaction between EF and CaM. In addition, we have carried out radioactive enzyme assays to study the affinity of MANT nucleotides for EF. MANT nucleotides are highly potent inhibitors of EF (Ki values in the nanomolar range). With the help of molecular modeling studies we have also observed that the positioning of the MANT group is very critical for the binding and inhibition of catalytic activity of EF. Our long‐term goal is to identify potent inhibitors for EF and explain the structural basis of inhibition of EF by X‐ray crystallography.