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Anthrax stealth molecule

PSI-SGKB [doi:10.1038/fa_psisgkb.2009.10]
Featured Article - March 2009
Short description: Bacillus anthracis relies on an unusual metabolite to evade human innate immunity and to be fully virulent in the host.Proc. Natl Acad. Sci. USA 105, 17133-17138 (2008)

Bacillus anthracis is a highly virulent microbe that causes the disease anthrax and has been used for bioterrorism. As part of its pathogenic armory, it produces an iron-chelating compound (a siderophore) called petrobactin, which is involved in ferric iron uptake by the bacterium and is essential for maximum virulence in mammals.

The petrobactin molecule is synthesized from citrate, spermidine and an unusual 3,4-isomer of dihydroxybenzoic acid, 3,4-DHBA, which forms the iron-chelating moiety. The 2,3-DHBA isomer is more common in bacterial siderophores, but its action can be blocked by the human innate immune system. Humans produce a protein, siderocalin, that can sequester 2,3-DHBA, thus preventing iron uptake by pathogens.

Unfortunately, siderocalin does not act on 3,4-DHBA, which gives B. anthracis a growth advantage over other pathogens. So, understanding how 3,4-DHBA is synthesized might reveal a weakness that could be exploited by antimicrobial drugs.

The asbF gene of B. anthracis had previously been shown to be essential for 3,4-DHBA synthesis. Extending their previous work, Pfleger et al. 1 , in collaboration with PSI MCSG [], now identify the enzymatic activity of the AsbF protein as a 3-dehydroshikimate (DHS) dehydratase and show that it converts the common bacterial metabolite 3-DHS to 3,4-DHBA. To produce the necessary amounts of AsbF protein to identify its activity and determine its structure, they expressed the B. anthracis asbF gene in Escherichia coli.

Pfleger et al. show that the reaction product of purified recombinant AbsF acting on the candidate substrate 3-DHS has an absorbance maximum at 290 nm, which corresponds to a DHBA chromophore, and confirmed this by mass spectrometry. Biochemical analysis revealed that the AbsF-catalyzed reaction was inhibited by the metal chelator EDTA, and fluorescence scanning of crystallized recombinant AbsF indicated that manganese is the predominant enzyme-bound metal.

The team then solved the crystal structure of AbsF bound to 3,4-DHBA. The structure is a (β/α)8-barrel (TIM) barrel; its N terminus is partially buried at the bottom part of the barrel and the C terminus is completely exposed to the solvent-filled channel. The researcher found that 3,4-DHBA is bound in the active site of AsbF, surrounded by several aromatic amino acid residues.

Structurally, AbsF belongs to the AP endonuclease 2 TIM barrel protein family and its closest homologs are xylose isomerase and myoinositol catabolism protein IoII. TIM barrel enzymes vary in the location of the metal-binding sites and the type of metal involved. In the case of AsbF, a manganese ion is bound to six atoms — five in the enzyme and one in 3,4-DHBA — that might contribute to catalysis by stabilizing an intermediate ligand or product.

Analysis of AsbF site-directed mutants, together with information from the structure, support a catalytic mechanism with an enolate intermediate, specifically an E1CB (elimination unimolecular via conjugate base) mechanism. AbsF uses a base to abstract the axial proton of 3-DHS from its adjacent aliphatic carbon atom (C4).

This work indicates that AsbF is a potential new target for inhibitors that might prevent B. anthracis infection. If petrobactin were disabled, B. anthracis would be able to use only its other siderophore, the 2,3-DHBA-containing bacillibactin, for iron uptake, a process that would be highly compromised by sequestration of bacillibactin by host siderocalin.

Maria Hodges


  1. B, F. Pfleger et al. Structural and functional analysis of AsbF: Origin of stealth 3,4-dihydroxybenzoic acid subunit for petrobactin biosynthesis.
    Proc. Natl Acad. Sci. USA 105, 17133-17138 (2008).

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