PMID:23029200

Citation	Kudryashova, E, Kalda, C and Kudryashov, DS (2012) Glutamyl phosphate is an activated intermediate in actin crosslinking by actin crosslinking domain (ACD) toxin. PLoS ONE 7:e45721
Abstract	Actin Crosslinking Domain (ACD) is produced by several life-threatening Gram-negative pathogenic bacteria as part of larger toxins and delivered into the cytoplasm of eukaryotic host cells via Type I or Type VI secretion systems. Upon delivery, ACD disrupts the actin cytoskeleton by catalyzing intermolecular amide bond formation between E270 and K50 residues of actin, leading to the formation of polymerization-deficient actin oligomers. Ultimately, accumulation of the crosslinked oligomers results in structural and functional failure of the actin cytoskeleton in affected cells. In the present work, we advanced in our understanding of the ACD catalytic mechanism by discovering that the enzyme transfers the gamma-phosphoryl group of ATP to the E270 actin residue, resulting in the formation of an activated acyl phosphate intermediate. This intermediate is further hydrolyzed and the energy of hydrolysis is utilized for the formation of the amide bond between actin subunits. We also determined the pH optimum for the reaction and the kinetic parameters of ACD catalysis for its substrates, ATP and actin. ACD showed sigmoidal, non-Michaelis-Menten kinetics for actin (K(0.5) = 30 µM) reflecting involvement of two actin molecules in a single crosslinking event. We established that ACD can also utilize Mg(2+)-GTP to support crosslinking, but the kinetic parameters (K(M) = 8 µM and 50 µM for ATP and GTP, respectively) suggest that ATP is the primary substrate of ACD in vivo. The optimal pH for ACD activity was in the range of 7.0-9.0. The elucidated kinetic mechanism of ACD toxicity adds to understanding of complex network of host-pathogen interactions.
Links	PubMed PMC3448709 Online version:10.1371/journal.pone.0045721
Keywords	Actins/chemistry; Animals; Autoradiography; Bacterial Toxins/chemistry; Biocatalysis; Cross-Linking Reagents/chemistry; Glutamine/analogs & derivatives; Glutamine/chemistry; Hydrogen-Ion Concentration; Kinetics; Rabbits
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
VIBCH:Q9KS12	enables	GO:0005524: ATP binding	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete
VIBCH:Q9KS12	enables	GO:0000287: magnesium ion binding	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete
VIBCH:Q9KS12	involved_in	GO:0018153: isopeptide cross-linking via N6-(L-isoglutamyl)-L-lysine	ECO:0000314: direct assay evidence used in manual assertion		P		Seeded From UniProt	complete
VIBCH:Q9KS12	involved_in	GO:0030042: actin filament depolymerization	ECO:0000314: direct assay evidence used in manual assertion		P		Seeded From UniProt	complete
VIBCH:Q9KS12	enables	GO:0016881: acid-amino acid ligase activity	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete

References

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