W.G.B. Voorhorst

Molecular and biochemical characterization of an endo-beta-1,3- glucanase of the hyperthermophilic archaeon Pyrococcus furiosus.

We report here the first molecular characterization of an endo-β-1,3-glucanase from an archaeon.Pyrococcus furiosus is a hyperthermophilic archaeon that is capable of saccharolytic growth. The isolated lamA gene encodes an extracellular enzyme that shares homology with both endo-β-1,3- and endo-β-1,3-1,4-glucanases of the glycosyl hydrolase family 16. After deletion of the N-terminal leader sequence, alamA fragment encoding an active endo-β-1,3-glucanase was overexpressed in Escherichia coli using the T7-expression system. The purified P. furiosus endoglucanase has highest hydrolytic activity on the β-1,3-glucose polymer laminarin and has some hydrolytic activity on the β-1,3-1,4 glucose polymers lichenan and barley β-glucan. The enzyme is the most thermostable endo-β-1,3-glucanase described up to now; it has optimal activity at 100–105 °C. In the predicted active site of glycosyl hydrolases of family 16 that show predominantly endo-β-1,3-glucanase activity, an additional methionine residue is present. Deletion of this methionine did not change the substrate specificity of the endoglucanase, but it did cause a severe reduction in its catalytic activity, suggesting a structural role of this residue in constituting the active site. High performance liquid chromatography analysis showed in vitrohydrolysis of laminarin by the endo-β-1,3-glucanase proceeds more efficiently in combination with an exo-β-glycosidase from P. furiosus (CelB). This most probably reflects the physiological role of these enzymes: cooperation during growth of P. furiosus on β-glucans.

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°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 αS1-casein and synthetic peptides.

Molecular characterization of fervidolysin, a subtilisin-like serine protease from the thermophilic bacterium Fervidobacterium pennivorans

Abstract. The fls gene encoding fervidolysin, a keratin-degrading proteolytic enzyme from the thermophilic bacterium Fervidobacterium pennivorans, was isolated using degenerate primers combined with Southern hybridization and inverse polymerase chain reaction. Further sequence characterization demonstrated that the 2.1-kb fls gene encoded a 699-amino-acid preproenzyme showing high homology with the subtilisin family of the serine proteases. It was cloned into a pET9d vector, without its signal sequence, and expressed in Escherichia coli. The heterologously produced fervidolysin was purified by heat incubation followed by ion exchange chromatography and emerged in the soluble fraction as three distinct protein bands, as judged from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino-terminal-sequence analysis of these bands and their comparison with that determined from biochemically purified keratinase and its predicted protein sequence, identified them as a 73-kDa fervidolysin precursor, a 58-kDa mature fervidolysin, and a 14-kDa fervidolysin propeptide. Using site-directed mutagenesis, the active-site histidine residue at position 79 was replaced by an alanine residue. The resulting fervidolysin showed a single protein band corresponding in size to the 73-kDa fervidolysin precursor, indicating that its proteolytic cleavage resulted from an autoproteolytic process. Knowledge-based modeling experiments showed a distinctive binding region for subtilases, in which binding of the propeptide could take place prior to autoproteolysis. Assays using keratin and other proteinaceous substrates did not display fervidolysin activity, perhaps because of the tight binding of the propeptide in the substrate-binding site, where it could then function as an inhibitor.