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Research Themes Drug discovery

Anthrax evasion tactics

PSI-SGKB [doi:10.1038/fa_psisgkb.2009.38]
Featured Article - September 2009
Short description: The crystal structure of a crucial anthrax capsule enzyme will aid the search for new therapies.

Ribbon diagram of the CapD transpeptidase from Bacillus anthracis cap operon in complex with non-hydrolyzable substrate analogue. The enzyme attaches poly-γ-D-glutamic acid to peptidoglycan and forms a protective capsule on the surface of germinating bacilli. The capsule is essential for virulence during anthrax infection and can be visualized by India ink staining.

The bacterium responsible for the disease anthrax, Bacillus anthracis, is highly virulent and is often deadly. Various countries have tested and deployed this pathogen as a weapon throughout the twentieth century, and more recently it has been used for bioterrorism, with spores being sent through the US postal system.

When the bacterium exists as a spore it is incredibly difficult to destroy and it can persist for decades or even centuries. Once the spore enters the bloodstream, it germinates, replicates and spreads to every tissue in the body, thanks to its ability to evade detection by macrophages and granulocytes.

Bacillus anthracis outwits the immune system by using a thick capsule in which it encases itself in. This capsule is unusual in that it is not formed of polysaccharides, which are found on the surface of most bacterial pathogens, but is instead formed from a peptide poly-γ-D-glutamic acid or PDGA. This peptide is itself unusual in that it is a D isoform rather the L form found in most proteins.

Several genes are needed for capsule biogenesis, but the one that caught the eye of Wu et al. from the PSI MCSG and the University of Chicago was CapD, which sits on the surface of the cell. CapD interacts with PDGA and attaches this compound to the bacteria's cell wall through a transpeptidation reaction.

The sequence of CapD is similar to those of members of the γ-glutamyl transpeptidase (GGT) family, which catalyse the first step in glutathione degradation. However, CapD is specific for PDGA and does not catalyze reactions with the smaller glutathione substrate.

In their report, Wu et al. 1 describe the crystal structure of CapD with and without glutamate dipeptide, a non-hydrolysable analogue of its natural substrate, and compare it with CapD from other bacteria and other GGT family members to discover how it performs this unique reaction.

The overall structure of CapD contains two central β-sheets stacked with cluster of ten α-helices above it and eight α-helices below it. Together it forms a six-layer alpha/alpha/beta/beta/alpha/alpha sandwich, similar to the four-layer alpha/beta/beta/alpha sandwich seen in Ntn hydrolases. CapD has a large solvent-accessible groove in the middle of the molecule between the lower and upper domains. It has a remarkable, extended Y-shaped positive charge on the surface of the structure with the active site threonine 352 at the bottom of this cleft.

The differences in the ligand-bound and ligand-free forms suggest that upon substrate binding several α-helices move towards the Y-shaped groove, making the overall CapD structure more compact. Also, a loop region, Pro494 to Phe497, near the active site becomes more structured and becomes a single-turn helix upon substrate binding. Several side chains also change conformation.

In common with Ntn hydrolases, CapD undergoes self cleavage to form an acyl–enzyme intermediate; without cleavage a part of the amino acid sequence, known as the P segment, blocks the active site. In the CapD structure, a water molecule is visible within the active site, and this would be crucial for cleavage of an internal CapD peptide bond, lending support to the autocatalysis hypothesis of CapD activation.

CapD's substrate, PDGA, is larger than those of other members of the GGT family, and comparison of the CapD structure with other GGT family members suggest that they cannot accommodate PDGA because they have a longer loop that blocks access to the active site.

Surprisingly, CapD's active site is exposed and yet it cannot hydrolyse glutathione. This is likely to be because the Y-shaped cleft mediates substrate recognition. From the structure and from site-directed mutations, Wu et al. suggest that Thr372, Pro427, Gly428 and Gly429 activate Thr352 and stabilize an oxyanion hole. Modeling of the natural substrate, PDGA, within the active site suggests that Asn431, Arg432 and Arg520 position PDGA for catalysis.

Armed with this information, the search for inhibitors of CapD is underway 2 .

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References

  1. R. Wu et al. Crystal structure of Bacillus anthracis transpeptidase enzyme CapD.
    J. Biol. Chem. (2009). doi:10.1074/jbc.M109.019034

  2. S. Richter et al. Capsule anchoring in Bacillus anthracis occurs by a transpeptidation reaction that is inhibited by capsidin.
    Mol. Microbiol. 71, 404-420 (2009). doi:10.1111/j.1365-2958.2008.06533.x

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