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 post-translational fatty acylation of two internal lysine residues. HlyC, 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. HlyC was thought to serve as an internal protein acyltransferase and remained uncharacterized until now. 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 acyltransfer was assayed. Acyl-ACP was the obligate acyl donor. Acyltransfer was catalyzed by HlyC monomer, and an acyl-enzyme intermediate was detected and shown to catalyze the reverse reaction. The reaction mechanism was examined by steady state kinetics, and the nature of inhibitions by reaction products was determined. The kinetic mechanism of the internal protein acylation was compatible with an uni uni iso uni uni ping pong with isomerization of the F form of the enzyme. Clues to the chemical mechanism for the acyltransferase were elucidated by both chemical modification studies and site directed mutagenesis of the enzyme. Chemical modification experiments ruled out any critical cysteines, serines, and lysine residues, but suggested a role for histidine(s) and tyrosine(s) in acyltransferase function. In order to examine the function of specific residues and possibly corroborate the chemical findings, site directed mutagenesis studies of the acyltransferase were employed. Seventeen residues that were conserved among 13 different RTX toxin acyltransferases were individually mutated, and the respective HlyCs expressed, and characterized. Residues that were critical for acyltransferase function included Gly 11, His 23, Tyr 70, and Gly 85. As with chemical modification data, mutagenesis ruled out any conserved, essential, cysteines or serines critical for HlyC acyltransferase activity.
| Identifer | oai:union.ndltd.org:ETSU/oai:dc.etsu.edu:etd-4376 |
| Date | 01 December 1998 |
| Creators | Trent, Michael S. |
| Publisher | Digital Commons @ East Tennessee State University |
| Source Sets | East Tennessee State University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Electronic Theses and Dissertations |
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