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

• Published in: Biochemistry (Easton) Vol. 38; no. 11; pp. 3433 - 3439
• Main Authors: Trent, M. Stephen, Worsham, Lesa M. S, Ernst-Fonberg, M. Lou
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 16.03.1999
• Subjects: Acetyltransferases - antagonists & inhibitors; Acetyltransferases - genetics; Acetyltransferases - metabolism; Acetyltransferases - physiology; Acyltransferases; Amino Acid Sequence; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Bacterial Proteins - pharmacology; Bacterial Proteins - physiology; Bacterial Toxins - genetics; Bacterial Toxins - metabolism; Catalysis; Conserved Sequence - genetics; Cysteine - genetics; Enzyme Activation - genetics; Escherichia coli; Escherichia coli - enzymology; Escherichia coli Proteins; Hemolysin Proteins - genetics; Hemolysin Proteins - metabolism; Hemolysin Proteins - pharmacology; Hemolysin Proteins - physiology; Histidine; Molecular Sequence Data; Mutagenesis, Site-Directed; Peptides - genetics; Repetitive Sequences, Amino Acid; Serine - genetics
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi982491u

HlyC is an internal protein acyltransferase that activates hemolysin, a toxic protein produced by pathogenic Escherichia coli. Acyl−acyl carrier protein (ACP) is the essential acyl donor. Separately subcloned, expressed, and purified prohemolysin A (proHlyA), HlyC, and [1-14C]myristoyl-ACP have been 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-negative 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 that neither the single conserved cysteine nor the three conserved serine residues were required for enzyme activity. Treatment with the reversible histidine-modifying diethyl pyrocarbonate (DEPC) inhibited acyltransferase activity, and acyltransferase activity was restored following hydroxylamine treatment. The substrate myristoyl-ACP protected HlyC from DEPC inhibition. These findings and spectral absorbance changes suggested that histidine, particularly a histidine proximal to the substrate binding site, was essential for enzyme activity. Site-directed mutageneses of the single conserved histidine residue, His23, to alanine, cysteine, or serine resulted in each instance in complete inactivation of the enzyme.