Characterization of the celB gene coding for beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus and its expression and site-directed mutation in Escherichia coli

Abstract

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...

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