The biochemistry of hemolysin toxin activation: characterization of HlyC, an internal protein acyltransferase
Trent, M.S.; Worsham, L.M.; Ernst-Fonberg, M.L.
Biochemistry 37(13): 4644-4652
ISSN/ISBN: 0006-2960 PMID: 9521785 DOI: 10.1021/bi971588y
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. Km and Vmax for HlyA were 0.94 microM and 7.5 pmol of acyl group transferred/min, respectively; Km and Vmax for myristoyl-ACP were 0.48 microM and 6.9 pmol/min. The kinetic parameters of different acyl-ACPs resembled a competitive inhibition as acyl group carbon chain length increased; Km's increased while Vmax'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.