期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2015
卷号：112
期号：23
页码：E3058-E3066
DOI：10.1073/pnas.1503832112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceSecreted pore-forming toxins of pathogenic bacteria such as Escherichia coli and Bordetella pertussis insert into cell membranes to subvert signaling and cause cell death, facilitating infection of human and animal hosts. These toxins require a unique activation step before secretion, the covalent linkage of lipid groups to specific lysines of the inactive protoxin, directed by a specialized toxin-activating acyl transferase (TAAT). Here, we present the TAAT crystal structure, the soluble dimeric topology, and likely active site, revealing that despite no discernible sequence similarity, TAATs are a structurally and functionally distinct group of the Gcn5-like N-acetyl transferase (GNAT) superfamily of modifying enzymes. Our findings open the way to further understanding of the unique toxin activation, and the possibility of inhibiting toxin action. Secreted pore-forming toxins of pathogenic Gram-negative bacteria such as Escherichia coli hemolysin (HlyA) insert into host-cell membranes to subvert signal transduction and induce apoptosis and cell lysis. Unusually, these toxins are synthesized in an inactive form that requires posttranslational activation in the bacterial cytosol. We have previously shown that the activation mechanism is an acylation event directed by a specialized acyl-transferase that uses acyl carrier protein (ACP) to covalently link fatty acids, via an amide bond, to specific internal lysine residues of the protoxin. We now reveal the 2.15-[IMG]f1.gif" ALT="A" BORDER="0"> resolution X-ray structure of the 172-aa ApxC, a toxin-activating acyl-transferase (TAAT) from pathogenic Actinobacillus pleuropneumoniae. This determination shows that bacterial TAATs are a structurally homologous family that, despite indiscernible sequence similarity, form a distinct branch of the Gcn5-like N-acetyl transferase (GNAT) superfamily of enzymes that typically use acyl-CoA to modify diverse bacterial, archaeal, and eukaryotic substrates. A combination of structural analysis, small angle X-ray scattering, mutagenesis, and cross-linking defined the solution state of TAATs, with intermonomer interactions mediated by an N-terminal -helix. Superposition of ApxC with substrate-bound GNATs, and assay of toxin activation and binding of acyl-ACP and protoxin peptide substrates by mutated ApxC variants, indicates the enzyme active site to be a deep surface groove.
