The biochemistry of hemolysin toxin activation: characterization of HlyC, an internal protein acyltransferase.
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.
Trent
M S
MS
Department of Biochemistry and Molecular Biology, James H. Quillen College of Medicine, Box 70 581, East Tennessee State University, Johnson City, Tennessee 37614, USA.
Worsham
L M
LM
Ernst-Fonberg
M L
ML
D000213:Acyl Carrier Protein / Q000378:metabolism; D000217:Acyltransferases; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001427:Bacterial Toxins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015153:Blotting, Western; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006461:Hemolysis; D006801:Humans; D007700:Kinetics; D011498:Protein Precursors / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
D000213:Acyl Carrier Protein; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins; C036507:Hlya protein, E coli; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D000217:Acyltransferases; C092237:HlyC protein, E coli