Analysis of the function of a hyperthermophilic endoglucanase from Pyrococcus horikoshii that hydrolyzes crystalline cellulose.
A hyperthermophilic beta-1,4 endoglucanase was identified in Pyrococcus horikoshii, a hyperthermophilic archaeon. In order to clarify the function of the protein in detail, structural and catalytic site studies were performed using protein engineering. By removing some of the C-terminal sequence of the ORF of the endoglucanase (PH1171), two types of recombinant proteins were expressed from one ORF, using Escherichia coli. One exhibited endoglucanase activity, and the other did not. An SD-like sequence was identified in the ORF of the endoglucanase. By removing the SD-like sequence without changing the amino acid sequence of the endoglucanase, one recombinant endoglucanase was prepared effectively from E. coli. From the analysis of the N- and C-terminal regions of the ORF, this endoglucanase appears to be a secreted and membrane-binding enzyme of P. horikoshii. A mutation analysis of the endoglucanase, using the synthetic substrate, indicated that Glu342 is a candidate for the active center and plays a critical role in the activity of the enzyme. Additional catalytic amino acid residues were not found. These results indicate that the catalytic residue of the enzyme is different from that of typical family 5 endoglucanase, even though it has a high homology to the endoglucanase from Acidothermus celluloliticus. The activity of the enzyme, using carboxy methylcellulose and crystalline cellulose as the substrates, was increased, but not for a synthetic low-molecular substrate when a carbohydrate-binding module of chitinase from P. furiosus was added to the C-terminal region.
Kashima
Yasuhiro
Y
Research Institute of Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST, Kansai), 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, Japan.
Mori
Kazushige
K
Fukada
Harumi
H
Ishikawa
Kazuhiko
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D002152:Calorimetry, Differential Scanning; D002384:Catalysis; D020134:Catalytic Domain; D002480:Cellulase / Q000737:chemistry; D002482:Cellulose / Q000737:chemistry*; D002688:Chitinases / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D004252:DNA Mutational Analysis; D017931:DNA Primers / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D016366:Open Reading Frames; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D043902:Pyrococcus horikoshii / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D017931:DNA Primers; D004220:Disulfides; D011994:Recombinant Proteins; D002482:Cellulose; D002688:Chitinases; D002480:Cellulase; D003545:Cysteine