A CATALYTIC TRIAD IS REQUIRED BY THE NONHEME HALOPEROXIDASES TO PERFORM HALOGENATION

The bacterial non-heme haloperoxidases are highly related to an estera se from Pseudomonas fluorescens, at structural and functional levels. Both types of enzymes displayed brominating activity and esterase acti vity. The presence of the serine-hydrolase motif Gly-X-Ser-X-Gly, in t he esterase as well as in all aligned haloperoxidase sequences, strong ly suggested that they belong to the serine-hydrolase family. Sequence alignment with several serine-hydrolases and secondary structure supe rimposition revealed the striking conservation of structural features characterising the alpha/beta-hydrolase fold structure in all halopero xidases. These structural predictions allowed us to identify a potenti al catalytic triad in haloperoxidases, perfectly matching the triad of all aligned serine-hydrolases. The structurally equivalent triad in t he chloroperoxidase CPO-P comprised the amino acids Serine 97, Asparti c acid 229 and Histidine 258. The involvement of this catalytic triad in halogenation was further assessed by inhibition studies and site-di rected mutagenesis. Inactivation of CPO-P by PMSF and DEPC strongly su ggested that the serine residue from the serine-hydrolase motif and an histidine residue are essential for halogenation, similar to that dem onstrated for typical serine-hydrolases. By site-directed mutagenesis of CPO-P, Ser-97 was exchanged against alanine or cysteine, Asp-229 ag ainst alanine and His-258 against glutamine. Western blot analysis ind icated that each mutant gene was efficiently expressed. Whereas the mu tant S97C conserved a very low residual activity, each other mutant S9 7A, D229A or H258Q was totally inactive. This study gives the direct d emonstration of the requirement of a catalytic triad in the halogenati on mechanism.