We report here the first molecular characterization of an endo-␤-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-␤-1,3-and endo-␤-1,3-1,4-glucanases of the glycosyl hydrolase family 16. After deletion of the N-terminal leader sequence, a lamA fragment encoding an active endo-␤-1,3-glucanase was overexpressed in Escherichia coli using the T7-expression system. The purified P. furiosus endoglucanase has highest hydrolytic activity on the ␤-1,3-glucose polymer laminarin and has some hydrolytic activity on the ␤-1,3-1,4 glucose polymers lichenan and barley ␤-glucan. The enzyme is the most thermostable endo-␤-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-␤-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 vitro hydrolysis of laminarin by the endo-␤-1,3-glucanase proceeds more efficiently in combination with an exo-␤-glycosidase from P. furiosus (CelB). This most probably reflects the physiological role of these enzymes: cooperation during growth of P. furiosus on ␤-glucans.

The celB gene encoding the cellobiose-hydrolyzing enzyme ␤-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus has been identified, cloned, and sequenced. The transcription and translation initiation sites of the celB gene have been determined, and archaeal control sequences were identified. The celB gene was overexpressed in Escherichia coli, resulting in high-level (up to 20% of total protein) production of ␤-glucosidase that could be purified by a two-step purification procedure. The ␤-glucosidase produced by E. coli had kinetic and stability properties similar to those of the ␤-glucosidase purified from P. furiosus. The deduced amino acid sequence of CelB showed high similarity with those of ␤-glycosidases that belong to glycosyl hydrolase family 1, implicating a conserved structure. Replacement of the conserved glutamate 372 in the P. furiosus ␤-glucosidase by an aspartate or a glutamine led to a high reduction in specific activity (200-or 1,000-fold, respectively), indicating that this residue is the active site nucleophile involved in catalysis above 100؇C.The most extensively studied representative of the hyperthermophilic organisms that have an optimum growth temperature above 85ЊC is Pyrococcus furiosus (12). P. furiosus is able to grow on a wide range of substrates, including complex polymers such as starch, glycogen, peptone, and casein or simple carbon compounds like cellobiose, maltose, and pyruvate (12,20,33). The main fermentation products are CO 2 and H 2 or alanine, the latter acting as an alternative electron sink (22). The disaccharides cellobiose and maltose are hydrolyzed by intracellular glucosidases (5, 20). The generated glucose was proposed to be further metabolized via a nonphosphorylated Entner-Doudoroff pathway (27, 33). However, recently it was discovered that sugars are fermented by P. furiosus via an Embden-Meyerhof pathway that involves two ADP-dependent kinases (19).Characterization of proteins from hyperthermophiles revealed that they are extremely thermostable and may have an optimum temperature of catalysis that exceeds the maximum growth temperature of their host (1,18). In addition to their remarkable thermostability, proteins from hyperthermophiles are often found to be highly resistant to chemical denaturation and to degradation by proteases (13). One of the most thermostable enzymes identified up to now is the ␤-glucosidase from P. furiosus, with a half-life of 85 h at 100ЊC (20). During growth on cellobiose, ␤-glucosidase can make up to 5% of the total cell protein of P. furiosus and is involved in the hydrolysis of the ␤-1,4-glycosidic bond between the two glucose moieties of the disaccharide (20). In addition, ␤-glucosidases constitute a group of well-studied enzymes among members of all three domains of life, Eucarya, Bacteria, and Archaea (16,17,35). Therefore, the pyrococcal ␤-glucosidase is a suitable model enzyme for the molecular characterization of structure-function relations of hyperthermostable enzymes. To study these relations by protein eng...

Desulfoferrodoxin from Desulfovibrio vulgaris, strain Hildenborough, is a homodimer of 28 kDa; it contains two Fe atoms per 14.0 kDa subunit. The N-terminal amino-acid sequence is homogeneous and corresponds to the previously described Rbo gene, which encodes a highly charged 14 kDa polypeptide without a leader sequence. Although one of the two iron centers, Fe,, has previously been described as a 'strained rubredoxin-like' site, EPR of the ferric form proves very similar to that of the pentagonal bipyramidally coordinated iron in ferric complexes of DTPA, diethylenetriaminepentaacetic acid: both systems have spin S = 5/2 and rhombicity E/D = 0.08. Unlike the Fe site in rubredoxin the FeA site in desulfoferrodoxin has a pH dependent midpoint potential with pK,, = 9.2 and pK, = 5.3. Upon reduction (Em,75 = +2 mV) Fe, exhibits an unusually sharp S = 2 resonance in parallel-mode EPR. The second iron, Fe,, has S = 5/2 and E/D = 0.33; upon reduction (E,,,7,5 = +90 mV) Fe, turns EPR-silent.

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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 NH 2 -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 NH 2 -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 ␣ S1 -casein and synthetic peptides.