title: Analysis of the putative substrate binding region of hyperthermophilic endoglucanase from Pyrococcus horikoshii.
abstract: A hyperthermophophilic beta-1,4 endoglucanase (family 5, cellulase) was identified in a hyperthermophilic archaeon Pyrococcus horikoshii and found to be capable of hydrolyzing crystalline cellulose at high temperatures. This hyperthermophilic enzyme has promise for applications in biomass utilization, but we have no information regarding the catalytic mechanism or structure of the enzyme. To determine its catalytic mechanism, we examined the roles of amino acids located in a loop near the speculative active site by the alanine scanning method. Ten mutants of the enzyme were constructed and expressed in Escherichia coli. The purified mutant enzymes were assayed for their hydrolytic activities on p-nitrophenyl cellobiose (pNG2), carboxylmethyl cellulose, and avicel. The results showed that His155, Arg156, and Ile162 play an important role in pNG2 binding capacity, and that H155 and I162 are important for catalysis.
authors.affiliation: National Institute of Advanced Industrial Science and Technology (AIST), Japan.
authors.forename: Han-Woo
authors.initials: HW
authors.lastname: Kim
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Takagi
authors.forename: Yoshihisa
authors.initials: Y
authors.lastname: Hagihara
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Ishikawa
mesh_terms: D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D001120:Arginine / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D002480:Cellulase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D006639:Histidine / Q000378:metabolism; D006868:Hydrolysis; D007532:Isoleucine / Q000378:metabolism; D007700:Kinetics; D009154:Mutation; D011485:Protein Binding; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D043902:Pyrococcus horikoshii / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D019843:Archaeal Proteins; D007532:Isoleucine; D006639:Histidine; D001120:Arginine; D002480:Cellulase