Hemolysin Toxin Activation, an Internal Fatty Acylation

Document Type

Article

Publication Date

12-1-1997

Description

Escherichia coli hemolysin (HlyA) is synthesized as nontoxic proHlyA. It is post-translationally modified by the internal protein transacylase HlyC to form toxic HtyA. The enzyme transfers a long chain fatty acid from acyl-acyl carrier protein (acyl-ACP) to one or two internal lysine residues forming an amide bond. HlyA is secreted and forms cation-specific channels in target cells leading to osmotic lysis. Acylation is the single factor that renders HlyA toxic. Previously erythrocyte hemolysis was used to monitor the function of the enzyme. A direct assay was developed in our laboratory using acyl-ACPs with radiolabelled acyl groups which permitted quantification of transacylase activity. Examination of diverse acyl-ACPs revealed that myristoyl-ACP was the preferred fatty acyt donor. CoA esters did not serve as substrates, ACPSIt (non-acylated ACP) was inhibitory. The different acyl-toxins generated in the direct assay were monitored for erythrocyte lysis, and the efficacy of the various acyl-ACPs as substrates differed from the lytic abilities of the corresponding acyl-toxins that were produced. Notably, an aeyl-enzyme intermediate was detected and characterized. Denaturing polyacrylamide gel electrophoresis of the acyl-enzyme intermediate, size exclusion chromatography, and chemical cross linking experiments between HlyC and acyl-ACP all indicated that the active form of the enzyme was a monomer. HlyC does not resemble any other known transacylase, and some confusion exists regarding its role in the fatty acylation of proHlyA. We have purified HlyC and demonstrated that the pure protein has transacylase activity; thus HlyC is the enzyme.

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