A catalytic triad is required by the non-heme haloperoxidases to perform halogenation.
The bacterial non-heme haloperoxidases are highly related to an esterase from Pseudomonas fluorescens, at structural and functional levels. Both types of enzymes displayed brominating activity and esterase activity. The presence of the serine-hydrolase motif Gly-X-Ser-X-Gly, in the esterase as well as in all aligned haloperoxidase sequences, strongly suggested that they belong to the serine-hydrolase family. Sequence alignment with several serine-hydrolases and secondary structure superimposition revealed the striking conservation of structural features characterising the alpha/beta-hydrolase fold structure in all haloperoxidases. These structural predictions allowed us to identify a potential catalytic triad in haloperoxidases, perfectly matching the triad of all aligned serine-hydrolases. The structurally equivalent triad in the chloroperoxidase CPO-P comprised the amino acids Serine 97, Aspartic acid 229 and Histidine 258. The involvement of this catalytic triad in halogenation was further assessed by inhibition studies and site-directed mutagenesis. Inactivation of CPO-P by PMSF and DEPC strongly suggested that the serine residue from the serine-hydrolase motif and an histidine residue are essential for halogenation, similar to that demonstrated for typical serine-hydrolases. By site-directed mutagenesis of CPO-P, Ser-97 was exchanged against alanine or cysteine, Asp-229 against alanine and His-258 against glutamine. Western blot analysis indicated that each mutant gene was efficiently expressed. Whereas the mutant S97C conserved a very low residual activity, each other mutant S97A, D229A or H258Q was totally inactive. This study gives the direct demonstration of the requirement of a catalytic triad in the halogenation mechanism.
Pelletier
I
I
Institut für Industrielle Genetik, Universität Stuttgart, Germany.
Altenbuchner
J
J
Mattes
R
R
D000595:Amino Acid Sequence; D001483:Base Sequence; D002711:Chloride Peroxidase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D007453:Iodide Peroxidase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010544:Peroxidases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D011480:Protease Inhibitors / Q000494:pharmacology; D017433:Protein Structure, Secondary; D011549:Pseudomonas / Q000201:enzymology*; D016415:Sequence Alignment; D014105:Tosyl Compounds / Q000494:pharmacology
D011480:Protease Inhibitors; D014105:Tosyl Compounds; C003614:4-toluenesulfonyl fluoride; D010544:Peroxidases; C026195:bromide peroxidase; D002711:Chloride Peroxidase; D007453:Iodide Peroxidase; D004047:Diethyl Pyrocarbonate