HlyC, the internal protein acyltransferase that activates hemolysin toxin:Role of conserved histidine, serine, and cysteine residues in enzymatic activity as probed by chemical modification and site-directed mutagenesis

HlyC is an internal protein acyltransferase that activates hemolysin, a tox ic protein produced by pathogenic Escherichia coli. Acyl-acyl carrier prote in (ACP) is the essential acyl donor. Separately subcloned, expressed, and purified prohemolysin A (proHlyA), HlyC, and [1-C-14]myristoyl-ACP have bee n used to study the conversion of proHlyA to HlyA [Trent, M. S., Worsham, L . M., and Ernst-Fonberg, M. L. (1998) Biochemistry 37, 4644-4655]. HlyC and hemolysin belong to a family of at least 13 toxins produced by Gram-negati ve bacteria. The homologous acyltransferases of the family show a number of conserved residues that are possible candidates for participation in acyl transfer. Specific chemical reagents and site-directed mutagenesis showed t hat neither the single conserved cysteine nor the three conserved serine re sidues were required for enzyme activity. Treatment with the reversible his tidine-modifying diethyl pyrocarbonate (DEPC) inhibited acyltransferase act ivity, and acyltransferase activity was restored following hydroxylamine tr eatment. The substrate myristoyl-ACP protected HlyC from DEPC inhibition. T hese findings and spectral absorbance changes suggested that histidine, par ticularly a histidine proximal to the substrate binding site, was essential for enzyme activity. Site-directed mutageneses of the single conserved his tidine residue, His23, to alanine, cysteine, or serine resulted in each ins tance in complete inactivation of the enzyme.