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 hyperthermophilic 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 immunological cross-reaction and identical NH2-terminal sequences in which the third 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 as a consequence of COOH-terminal autoproteolysis. The 4194-base pair pls gene encoding pyrolysin was isolated and characterized, and its transcription initiation site was identified. The deduced pyrolysin sequence indicated a prepro-enzyme organization, with a 1249-residue mature protein composed of an NH2-terminal catalytic domain with considerable homology to subtilisin-like serine proteases and a COOH-terminal domain that contained most of the 32 possible N-glycosylation sites. The archaeal pyrolysin showed highest homology with eucaryal tripeptidyl peptidases II on the amino acid level but a different cleavage specificity as shown by its endopeptidase activity toward caseins, casein fragments including alphaS1-casein and synthetic peptides.
Voorhorst
W G
WG
Department of Microbiology, Wageningen Agricultural University, 6703 CT Wageningen.
Eggen
R I
RI
Geerling
A C
AC
Platteeuw
C
C
Siezen
R J
RJ
Vos
W M
WM
D000595:Amino Acid Sequence; D001105:Archaea / Q000201:enzymology*; D019843:Archaeal Proteins; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D004792:Enzyme Precursors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D005798:Genes, Bacterial; D006358:Hot Temperature; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011498:Protein Precursors; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013381:Subtilisins / Q000737:chemistry; D014158:Transcription, Genetic
D019843:Archaeal Proteins; D017931:DNA Primers; D004792:Enzyme Precursors; D011498:Protein Precursors; D012697:Serine Endopeptidases; D013381:Subtilisins; C101133:pls protein, Pyrococcus furiosus