Analysis of the function of a hyperthermophilic endoglucanase from Pyrococcus horikoshii that hydrolyzes crystalline cellulose

Extremophiles : Life Under Extreme Conditions
Yasuhiro KashimaKazuhiko Ishikawa

Abstract

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 t...Continue Reading

Citations

Nov 8, 2008·Protein Engineering, Design & Selection : PEDS·P G BagosS J Hamodrakas
Dec 5, 2008·Acta Crystallographica. Section F, Structural Biology and Crystallization Communications·Han-Woo KimKazuhiko Ishikawa
Dec 6, 2011·Acta Crystallographica. Section F, Structural Biology and Crystallization Communications·Yuji KadoKazuhiko Ishikawa
Mar 24, 2012·Acta Crystallographica. Section F, Structural Biology and Crystallization Communications·Misumi KataokaKazuhiko Ishikawa
Jun 25, 2013·Applied and Environmental Microbiology·Hirokazu SuzukiToshihisa Ohshima
Jul 25, 2006·Bioscience, Biotechnology, and Biochemistry·Takashi Oku, Kazuhiko Ishikawa
Oct 12, 2007·Bioscience, Biotechnology, and Biochemistry·Han-Woo KimKazuhiko Ishikawa
Mar 4, 2009·Acta Crystallographica. Section F, Structural Biology and Crystallization Communications·Baisong ZhengZhiyong Lou
Oct 31, 2006·Extremophiles : Life Under Extreme Conditions·Hee-Jin KangKazuhiko Ishikawa
Jul 24, 2008·Metabolic Engineering·Chung-Jung ChouRobert M Kelly
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Oct 23, 2009·Proteins·Han-Woo Kim, Kazuhiko Ishikawa
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Mar 7, 2014·Acta Crystallographica. Section D, Biological Crystallography·Makoto NakabayashiKazuhiko Ishikawa
Sep 12, 2014·Bioscience, Biotechnology, and Biochemistry·Misumi Kataoka, Kazuhiko Ishikawa

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