Characterization of a novel carboxypeptidase produced by the entomopathogenic fungus Metarhizium anisopliae.
Preparative isoelectric focusing and gel filtration chromatography were used to purify a carboxypeptidase produced by the entomopathogenic fungus Metarhizium anisopliae during growth on cockroach cuticle. The enzyme was inhibited by diisopropyl fluorophosphate, implying involvement of a serine residue in catalysis. However, the M. anisopliae enzyme differed from most serine carboxypeptidases in also being inhibited by 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 properties resemble those exhibited by some metalloproteases but the enzyme is not inhibited by Cd2+; nor do Zn2+ or Co2+ restore activity in enzyme inhibited with phenanthroline. The amino-terminal sequence (22 residues) showed no similarity to other protein sequences. Unlike previously reported fungal carboxypeptidases, the M. anisopliae enzyme is powerfully inhibited by potato carboxypeptidase inhibitor. The carboxypeptidase shows a broad primary specificity toward amino acids with hydrophobic side groups in a series of N-blocked dipeptides, with substrates with phenylalanine being the most rapidly hydrolyzed. The S1 subsite also accommodated Glu, confirming its low selectivity. Proline at P1 or P1 resulted in a very poor substrate. The specificity of the carboxypeptidase complements that of the subtilisin-like protease (Pr1) of M. anisopliae. Both Pr1 and the carboxypeptidase are produced during carbon and nitrogen deprivation, which indicates that the exopeptidase functions with Pr1 to degrade peptides to supply amino acids during starvation and pathogenicity.
St Leger
R J
RJ
Boyce Thompson Institute for Plant Research, Ithaca, New York 14853-1801.
Bidochka
M J
MJ
Roberts
D W
DW
D000595:Amino Acid Sequence; D000818:Animals; D002268:Carboxypeptidases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D002850:Chromatography, Gel; D003058:Cockroaches; D004151:Dipeptides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007525:Isoelectric Focusing; D007531:Isoflurophate / Q000494:pharmacology; D007700:Kinetics; D003904:Mitosporic Fungi / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009842:Oligopeptides / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011480:Protease Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity
D004151:Dipeptides; D009842:Oligopeptides; D010446:Peptide Fragments; D011480:Protease Inhibitors; D007531:Isoflurophate; D002268:Carboxypeptidases