HlyC, the internal protein acyltransferase that activates hemolysin toxin: roles of various conserved residues in enzymatic activity as probed by site-directed mutagenesis.
Hemolysin, a toxic protein produced by pathogenic Escherichia coli, is one of a family of homologous toxins and toxin-processing proteins produced by Gram-negative bacteria. HlyC, an internal protein acyltransferase, converts it from nontoxic prohemolysin to toxic hemolysin. Acyl-acyl carrier protein is the essential acyl donor. The acyltransferase reaction progresses through formation of a binary complex between acyl-ACP and HlyC to a reactive acyl-HlyC intermediate [Trent, M. S., Worsham, L. M., and Ernst-Fonberg, M. L. (1998) Biochemistry 37, 4644-4655]. The homologous acyltransferases of the family have a number of conserved amino acid residues that may be catalytically important. Experiments to illuminate the reaction mechanism were done. The formation of an acyl-enzyme intermediate suggested that the reaction likely proceeded through two partial reactions. The reversibility of the first partial reaction was shown by using separately subcloned, purified, and expressed substrates and enzyme. The effects of single site-directed mutations of conserved residues of HlyC on different portions of reaction progress (binary complex formation, acyl-enzyme formation, and enzyme activity, including kinetic parameters) were determined. Mutations of His23, the only residue essential for activity, formed normal binary complexes but were unable to form acyl-HlyC. The same was seen with S20A, a mutant with greatly impaired activity. Mutation of two conserved tyrosines separately to glycines results in greatly impaired binary complex and acyl-HlyC formation, but mutation of those residues to phenylalanines restored behavior to wild-type.
Department of Biochemistry and Molecular Biology, James H. Quillen College of Medicine, Box 70,581, East Tennessee State University, Johnson City, Tennessee 37614, USA.
M S
MS
Trent
L M
LM
Worsham
M L
ML
Ernst-Fonberg
D000213:Acyl Carrier Protein / Q000737:chemistry / Q000378:metabolism; D000217:Acyltransferases; D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001427:Bacterial Toxins / Q000378:metabolism*; D017124:Conserved Sequence; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000378:metabolism; D019281:Dimerization; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020449:Repetitive Sequences, Amino Acid; D017386:Sequence Homology, Amino Acid
D000213:Acyl Carrier Protein; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins; D000217:Acyltransferases; C092237:HlyC protein, E coli; D000409:Alanine