Analysis of aminopeptidase and dipeptidylpeptidase IV from the entomopathogenic fungus Metarhizium anisopliae

Abstract

Analytical and preparative isoelectric focusing were used to separate extracellular isoenzymes of aminopeptidase (pI 4.51, M(r) 45,000, pH optimum 7.0) and prolyl-dipeptidylpeptidase (pI 4.01, M(r) 74,000, pH optimum 8.0) produced by the entomopathogenic fungus Metarhizium anisopliae during growth on locust cuticle. Production of both activities is repressed by readily utilized nitrogen sources, but unlike the aminopeptidase, the dipeptidylpeptidase was also excreted at high levels during growth on casein. Casein-grown cultures contained additional isoenzymes with activity against lysyl-alanyl-4-methoxy-2-naphthylamine indicating M. anisopliae possesses multiple peptidases as an adaptation to different nutrient conditions. The aminopeptidase hydrolysed alanyl-leucyl-alanine and showed a broad specificity versus monoaminoacyl beta-naphthylamine (beta NA) substrates with alanine beta NA being the most rapidly hydrolysed. Inhibition by both bestatin and amastatin indicated similarities to the class of alanyl aminopeptidases (aminopeptidase M). Metal complexing agents also inhibited the aminopeptidase indicating a metal ion requirement. A specific inhibitor for serine proteases [diisopropyl fluorophosphate (DFP)] was without effect. The dipeptidylpeptidase showed a strong preference for substrates having a penultimate proline residue including alanyl-prolyl-glycine and aa-prolyl-beta NA substrates. The enzyme showed a broad specificity at the N-terminal amino acid. Inhibition by diprotin A indicates similarities with mammalian prolyl-dipeptidylpeptidases. The enzyme was also inhibited by DFP, implying involvement of a serine residue in catalysis. The results are discussed in the context of cuticle degradation and the participation of exopeptidases as mediators in releasing amino acids necessary for pathogen growth.