An investigation of the nature and function of module 10 in a family F/10 xylanase FXYN of Streptomyces olivaceoviridis E-86 by module shuffling with the Cex of Cellulomonas fimi and by site-directed mutagenesis

Satoshi Kaneko, Atsushi Kuno, Zui Fujimoto, Daisuke Shimizu, Sachiko MacHida, Yoko Sato, Kei Yura, Mitiko Go, Hiroshi Mizuno, Kazunari Taira, Isao Kusakabe, Kiyoshi Hayashi

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

Although the amino acid homology in the catalytic domain of FXYN xylanase from Streptomyces olivaceoviridis E-86 and Cex xylanase from Cellulomonas fimi is only 50%, an active chimeric enzyme was obtained by replacing module 10 in FXYN with module 10 from Cex. In the family F/10 xylanases, module 10 is an important region as it includes an acid/base catalyst and a substrate binding residue. In FXYN, module 10 consists of 15 amino acid residues, while in Cex it consists of 14 amino acid residues. The K(m) and k(cat) values of the chimeric xylanase FCF-C10 for PNP-xylobioside (PNP-X2) were 10-fold less than those for FXYN. CD spectral data indicated that the structure of the chimeric enzyme was similar to that of FXYN. Based on the comparison of the amino acid sequences of FXYN and Cex in module 10, we constructed four mutants of FXYN. When D133 or S135 of FXYN was deleted, the kinetic properties were not changed from those of FXYN. By deletion of both D133 and S135, the K(m) value for PNP-X2 decreased from the 2.0 mM of FXYN to 0.6 mM and the k(cat) value decreased from the 20 s-1 of FXYN to 8.7 s-1. Insertion of Q140 into the doubly deleted mutant further reduced the K(m) value to 0.3 mM and the k(cat) value to 3.8 s-1. These values are close to those for the chimeric enzyme FCF-C10. These results indicate that module 10 itself is able to accommodate changes in the sequence position of amino acids which are critical for enzyme function. Since changes of the spatial position of these amino acids would be expected to result in enzyme inactivation, module 10 must have some flexibility in its tertiary structure. The structure of module 10 itself also affects the substrate specificity of the enzyme. Copyright (C) 1999 Federation of European Biochemical Societies.

Original languageEnglish
Pages (from-to)61-66
Number of pages6
JournalFEBS Letters
Volume460
Issue number1
DOIs
Publication statusPublished - 1999 Oct 22

Keywords

  • Cellulomonas fimi
  • Cex
  • Chimeric xylanase
  • Family 10 xylanase
  • Module
  • Streptomyces olivaceoviridis

ASJC Scopus subject areas

  • Biophysics
  • Structural Biology
  • Biochemistry
  • Molecular Biology
  • Genetics
  • Cell Biology

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