Module shuffling of a family F/10 xylanase: Replacement of modules M4 and M5 of the FXYN of Streptomyces olivaceoviridis E-86 with those of the Cex of Cellulomonas fimi

Satoshi Kaneko, Shinnosuke Iwamatsu, Atsushi Kuno, Zui Fujimoto, Yoko Sato, Kei Yura, Mitiko Go, Hiroshi Mizuno, Kazunari Taira, Tsunemi Hasegawa, Isao Kusakabe, Kiyoshi Hayashi

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Abstract

To facilitate an understanding of structure-function relationships, chimeric xylanases were constructed by module shuffling between the catalytic domains of the FXYN from Streptomyces olivaceoviridis E-86 and the Cex from Cellulomonas fimi. In the family F/10 xylanases, the modules M4 and M5 relate to substrate binding so that modules M4 and M5 of the FXYN were replaced with those of the Cex and the chimeric enzymes denoted FCF-C4, FCF-C5 and FCF-C4,5 were constructed. The kcat value of FCF-C5 for p-nitrophenyl-β-D-cellobioside was similar to that of the FXYN (2.2 s-1); however, the kcat value of FCF-C4 for p-nitrophenyl-β-D-cellobioside was significantly higher (7.0 s-1). The loss of the hydrogen bond between E46 and S22 or the presence of the I49W mutation would be expected to change the position of Q88, which plays a pivotal role in discriminating between glucose and xylose, resulting in the increased kcat value observed for FCF-C4 acting on p-nitrophenyl-β-D-cellobioside since module M4 directly interacts with Q88. To investigate the synergistic effects of the different modules, module M10 of the FCF-C4 chimera was replaced with that of the Cex. The effects of replacement of module M4 and M10 were almost additive with regard to the Km and kcat values.

Original languageEnglish
Pages (from-to)873-879
Number of pages7
JournalProtein Engineering
Volume13
Issue number12
Publication statusPublished - 2000
Externally publishedYes

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Keywords

  • Cellulomonas fimi
  • Cex,chimeric xylanase
  • Family 10 xylanase
  • Module
  • Streptomyces olivaceoviridis

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Cite this

Kaneko, S., Iwamatsu, S., Kuno, A., Fujimoto, Z., Sato, Y., Yura, K., Go, M., Mizuno, H., Taira, K., Hasegawa, T., Kusakabe, I., & Hayashi, K. (2000). Module shuffling of a family F/10 xylanase: Replacement of modules M4 and M5 of the FXYN of Streptomyces olivaceoviridis E-86 with those of the Cex of Cellulomonas fimi. Protein Engineering, 13(12), 873-879.