The prolyl aminopeptidase from Lactobacillus delbrueckii subsp. bulgaricusbelongs to the alpha/beta hydrolase fold family

Prolyl aminopeptidase (PepIP) of Lactobacillus delbrueckii subsp. bulgaricu s displays the Gly-x-Ser-x-Gly-Cly consensus motif surrounding the catalyti c serine of the prolyl oligopeptidases family. Sequence comparison revealed that this motif and two other domains appear well conserved among bacteria l PepIPs and members of the alpha/beta hydrolase fold family. Secondary str uctural predictions of PepIP were performed from amino acid sequence and co rroborated by circular dichroism analysis. These predictions well matched t he core structure of alpha/beta hydrolases organised in eight beta-sheets c onnected by alpha-helices. We obtained 26 mutants of PepIP by chemical or s ite-directed mutagenesis. Most substitutions associated with stable and ina ctive mutant proteins were mainly located in the three conserved boxes (inc luding the catalytic serine motif). Taken together, our results strongly su ggest that PepIP belongs to the alpha/beta hydrolase fold family and that S er(107), Asp(246) and His(273) constitute the catalytic triad of the enzyme . (C) 1999 Elsevier Science B.V. All rights reserved.