Cloning, expression, and substrate specificity of MeCPA, a zinc carboxypeptidase that is secreted into infected tissues by the fungal entomopathogen Metarhizium anisopliae

To date zinc carboxypeptidases have only been found in animals and actinomy cete bacteria. A cDNA clone (MeCPA) for a novel fungal (Metarhizium anisopl iae) carboxypeptidase (MeCPA) was obtained by using reverse transcription d ifferential display polymerase chain reaction to identify pathogenicity gen es. MeCPA resembles pancreatic carboxypeptidases in being synthesized as a precursor species (418 amino acids) containing a large amino-terminal fragm ent (99 amino acids). The mature (secreted) form of MeCPA shows closest ami no acid identity to human carboxypeptidases Al (35%) and A2 (37%). MeCPA wa s expressed in an insect cell line yielding an enzyme with dual A1 + A2 spe cificity for branched aliphatic and aromatic COOH-terminal amino acids. How ever, in contrast to the very broad spectrum A + B-type bacterial enzymes, MeCPA lacks B-type activity against charged amino acids. This is predictabl e as key catalytic residues determining the specificity of MeCPA are conser ved with those of mammalian A-type carboxypeptidases. Thus, in evolutionary terms the fungal enzyme is an intermediate between the divergence of A and B forms and the differentiation of the A form into A1 and A2 isoforms, Ult rastructural immunocytochemistry of infected host (Manduca sexta) cuticle d emonstrated that MeCPA participates with the concurrently produced endoprot eases in procuring nutrients; an equivalent function to digestive pancreati c enzymes.