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 withthe Cex of Cellulomonas fimi and by site-directed mutagenesis

Although the amino acid homology in the catalytic domain of FXYN xylanase f rom Streptomyces olivaceoviridis E-86 and Cex xylanase from Cellulomonas fi mi 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 substrat e binding residue. In FXYN, module 10 consists of 15 amino acid residues, w hile in Cex it consists of 14 amino acid residues. The K-m and k(cat) value s of the chimeric xylanase FCF-C10 for PKP-xylobioside (PNP-X-2) were 10-fo ld 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 f our mutants of FXYN, When D133 or S135 of FXYN,vas deleted, the kinetic pro perties were not changed from those of FXYN. By deletion of both D133 and S 135, the K-m value for PNP-X-2 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). Inse rtion 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 thos e for the chimeric enzyme FCF-C10, These results indicate that module 10 it self is able to accommodate changes in the sequence position of amino acids which are critical for enzyme function, Since changes of the spatial posit ion of these amino acids would be expected to result in enzyme inactivation , module 10 must have some flexibility in its tertiary structure. The struc ture of module 10 itself also affects the substrate specificity of the enzy me. (C) 1999 Federation of European Biochemical Societies.