CHARACTERIZATION OF A NOVEL CARBOXYPEPTIDASE PRODUCED BY THE ENTOMOPATHOGENIC FUNGUS METARHIZIUM-ANISOPLIAE

Preparative isoelectric focusing and gel filtration chromatography wer e used to purify a carboxypeptidase produced by the entomopathogenic f ungus Metarhizium anisopliae during growth on cockroach cuticle. The e nzyme was inhibited by diisopropyl fluorophosphate, implying involveme nt of a serine residue in catalysis. However, the M. anisopliae enzyme differed from most serine carboxypeptidases in also being inhibited b y the metal chelator 1,10-phenanthroline and in being a small (30 kDa) , basic (pI 9.97) protein with a neutral pH optima (pH 6.8). These pro perties resemble those exhibited by some metalloproteases but the enzy me is not inhibited by Cd2+; nor do Zn2+ or Co2+ restore activity in e nzyme inhibited with phenanthroline. The amino-terminal sequence (22 r esidues) showed no similarity to other protein sequences. Unlike previ ously reported fungal carboxypeptidases, the M. anisopliae enzyme is p owerfully inhibited by potato carboxypeptidase inhibitor. The carboxyp eptidase shows a broad primary specificity toward amino acids with hyd rophobic side groups in a series of N-blocked dipeptides, with substra tes with phenylalanine being the most rapidly hydrolyzed. The S-1 subs ite also accommodated Glu, confirming its low selectivity. Proline at P-1 or P'(1) resulted in a very poor substrate. The specificity of the carboxypeptidase complements that of the subtilisin-like protease (Pr 1) of M. anisopliae. Both Pr1 and the carboxypeptidase are produced du ring carbon and nitrogen deprivation, which indicates that the exopept idase functions with Pr1 to degrade peptides to supply amino acids dur ing starvation and pathogenicity. (C) 1994 Academic Press, Inc.