The Biochemistry of Hemolysin Toxin Activation: Characterization of HlyC, an Internal Protein Acyltransferase
Document Type
Article
Publication Date
3-31-1998
Description
Hemolysin toxin produced and secreted by pathogenic Escherichia coli is one of a family of cytolytic, structurally homologous protein toxins known as RTX (repeats in toxin) toxins. RTX toxins are products of a gene cluster, CABD. The A gene product, nontoxic hemolysin (proHlyA), is made toxic by posttranslational fatty acylation of two internal lysine residues. HlyC, the C gene product, is essential for acylation, and acyl-acyl carrier protein (ACP) is the acyl donor. HlyB and HlyD are involved in secretion of the toxin. ProHlyA and HlyC were separately subcloned, expressed, and purified, and acyl-ACPs with diverse radioactive acyl groups were synthesized. With these proteins, the conversion of proHlyA to HlyA by acyl transfer was assayed. Acyl-ACP was the obligate acyl donor. Acyl transfer was catalyzed by HlyC monomer, and an acyl-enzyme intermediate was shown. Reaction was inhibited by ACPSH but not by fatty acid or fatty-acyl CoA. K(m) and V(max) for HlyA were 0.94 μM and 7.5 pmol of acyl group transferred/min, respectively; K(m) and V(max) for myristoyl-ACP were 0.48 μM and 6.9 pmol/min. The kinetic parameters of different acyl-ACPs resembled a competitive inhibition as acyl group carbon chain length increased; K(m)'s increased while V(max)'s remained unchanged. The different kinetic efficacies in the acyltransferase reaction of the ACPs with different acyl groups contrasted notably with the lytic powers of the corresponding acyl-toxins that they generated.
Citation Information
Trent, M. Stephen; Worsham, Lesa M.S.; and Ernst-Fonberg, M. Lou. 1998. The Biochemistry of Hemolysin Toxin Activation: Characterization of HlyC, an Internal Protein Acyltransferase. Biochemistry. Vol.37(13). 4644-4652. https://doi.org/10.1021/bi971588y PMID: 9521785 ISSN: 0006-2960