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Glutamyl Phosphate Is an Activated Intermediate in Actin Crosslinking by Actin Crosslinking Domain (ACD) Toxin
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 inter...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Public Library of Science
2012
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3448709/ https://www.ncbi.nlm.nih.gov/pubmed/23029200 http://dx.doi.org/10.1371/journal.pone.0045721 |
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author | Kudryashova, Elena Kalda, Caitlin Kudryashov, Dmitri S. |
author_facet | Kudryashova, Elena Kalda, Caitlin Kudryashov, Dmitri S. |
author_sort | Kudryashova, Elena |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-3448709 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2012 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-34487092012-10-01 Glutamyl Phosphate Is an Activated Intermediate in Actin Crosslinking by Actin Crosslinking Domain (ACD) Toxin Kudryashova, Elena Kalda, Caitlin Kudryashov, Dmitri S. PLoS One Research Article 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. Public Library of Science 2012-09-21 /pmc/articles/PMC3448709/ /pubmed/23029200 http://dx.doi.org/10.1371/journal.pone.0045721 Text en © 2012 Kudryashova et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Kudryashova, Elena Kalda, Caitlin Kudryashov, Dmitri S. Glutamyl Phosphate Is an Activated Intermediate in Actin Crosslinking by Actin Crosslinking Domain (ACD) Toxin |
title | Glutamyl Phosphate Is an Activated Intermediate in Actin Crosslinking by Actin Crosslinking Domain (ACD) Toxin |
title_full | Glutamyl Phosphate Is an Activated Intermediate in Actin Crosslinking by Actin Crosslinking Domain (ACD) Toxin |
title_fullStr | Glutamyl Phosphate Is an Activated Intermediate in Actin Crosslinking by Actin Crosslinking Domain (ACD) Toxin |
title_full_unstemmed | Glutamyl Phosphate Is an Activated Intermediate in Actin Crosslinking by Actin Crosslinking Domain (ACD) Toxin |
title_short | Glutamyl Phosphate Is an Activated Intermediate in Actin Crosslinking by Actin Crosslinking Domain (ACD) Toxin |
title_sort | glutamyl phosphate is an activated intermediate in actin crosslinking by actin crosslinking domain (acd) toxin |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3448709/ https://www.ncbi.nlm.nih.gov/pubmed/23029200 http://dx.doi.org/10.1371/journal.pone.0045721 |
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