ISOLATION AND CHARACTERIZATION OF THE HYPERTHERMOSTABLE SERINE-PROTEASE, PYROLYSIN, AND ITS GENE FROM THE HYPERTHERMOPHILIC ARCHAEON PYROCOCCUS-FURIOSUS

The hyperthermostable serine protease pyrolysin from the hyperthermoph ilic archaeon Pyrococcus furiosus was purified from membrane fractions . Two proteolytically active fractions were obtained, designated high (HMW) and low (LMW) molecular weight pyrolysin, that showed immunologi cal cross-reaction and identical NH2-terminal sequences in which the t hird residue could be glycosylated, The HMW pyrolysin showed a subunit mass of 150 kDa after acid denaturation. Incubation of HMW pyrolysin at 95 degrees C resulted in the formation of LMW pyrolysin, probably a s a consequence of COOH-terminal autoproteolysis. The 4194-base pair p ls gene encoding pyrolysin was isolated and characterized, and its tra nscription initiation site was identified. The deduced pyrolysin seque nce indicated a prepro-enzyme organization, with a 1249-residue mature protein composed of an NH2-terminal catalytic domain with considerabl e homology to subtilisin-like serine proteases and a COOH-terminal dom ain that contained most of the 32 possible N-glycosylation sites, The archaeal pyrolysin showed highest homology with eucaryal tripeptidyl p eptidases II on the amino acid level but a different cleavage specific ity as shown by its endopeptidase activity toward caseins, casein frag ments including alpha(S1)-casein and synthetic peptides.