ISOFORMS OF THE CUTICLE-DEGRADING PR1 PROTEINASE AND PRODUCTION OF A METALLOPROTEINASE BY METARHIZIUM-ANISOPLIAE

The entomopathogenic fungus, Metarhizium anisopliae, produces three di stinct types of proteinases during growth on cockroach cuticle. These were separated by analytical isoelectric focusing and characterized ac cording to their substrate specificity and inhibition patterns as Pr1 subtilisin-like proteinases (four isoforms pI range similar to 9.3-10. 2), a thermolysin-like metalloproteinase (pI similar to 7.3), and tryp sin-like serine Pr2 proteinases (two major isoforms, pI similar to 4.4 and 4.9 and two minor isoforms, pI similar to 5.2). Preparative isoel ectric focusing was used to separate the four Pr1(2) components produc ed during growth on cockroach cuticle with isoelectric points of 10.2 (m = 30.2 kDa), 9.8 (m = 28.5 kDa), 9.3 (m = 29.5 kDa), and 9.0 (m = 3 1.5 kDa). Two of the isoforms were also produced, at diminished levels , during growth on elastin or cellulose presumably as a result of carb on and nitrogen derepression. The pI 10.2 Pr1 differed from the other isoforms in preferring alanine over bulky hydrophobic groups at P-2 an d P-3 in discriminating against proline at P-2 and in its lack of sens itivity to tetra-butyloxycarbonyl-Gly-Leu-Phe-chlorcomethyl ketone. Di fferences in the N-terminal amino acid sequences confirmed that the fo ur isoforms are related products of at least two distinct genes. The i soforms showed similar primary specificities, with the aromatic P-1 ph enylalanine being 10- to 16-fold more reactive than a P-1 leucine resi due reflected principally in K-cat. However, methionine (containing a long unsubstituted side chain) was also a good substrate for each isof orm confirming the low selectivity of their S-1 subsites. The isoforms all degraded a variety of solubilized cuticle proteins, with high-mol ecular-weight acidic proteins being preferentially hydrolyzed. The met alloproteinase is active against the Pr1 substrate succinyl-(Ala)(2)-P ro-Phe-7-amino-4-coumarin trifluoromethyl, but differs from the Pr1 is oforms in being inhibited by 1,10-phenanthroline and phosphoramidon. T he potential role of the metalloproteinase in pathogenicity is discuss ed. (C) 1994 Academic Press, Inc.