Characterization of active-site aromatic residues in xylanase A from Streptomyces lividans.


Abstract

The role of four aromatic residues (W85, Y172, W266 and W274) in the structure-function relationship in xylanase A from Streptomyces lividans (XlnA) was investigated by site-directed mutagenesis where each residue was subjected to three substitutions (W85A/H/F; W266A/H/F; W274A/H/F and Y172A/F/S). These four amino acids are highly conserved among family 10 xylanases and structural data have implicated them in substrate binding at the active site. Far-UV circular dichroism spectroscopy was used to show that the overall structure of XlnA was not affected by any of these mutations. High-performance liquid chromatographic analysis of the hydrolysis products of birchwood xylan and xylopentaose showed that mutation of these aromatic residues did not alter the enzyme's mode of action. As expected, though, it did reduce the affinity of XlnA for birchwood xylan. A comparison of the kinetic parameters of different mutants at the same position demonstrated the importance of the aromatic nature of W85, Y172 and W274 in substrate binding. Replacement of these residues by a phenylalanine resulted in mutant proteins with a K(M) closer to that of the wild-type protein in comparison with the other mutations analyzed. The kinetic analysis of the mutant proteins at position W266 indicated that this amino acid is important for both substrate binding and efficient catalysis by XlnA. These studies also demonstrated the crucial role of these active site aromatic residues for the thermal stability of XlnA. Study holds ProTherm entries: 12868, 12869, 12870, 12871, 12872, 12873, 12874, 12876, 12878, 12879, 12880, 12881, 12882, 12883, 12884, 12885, 12886, 12887, 12888, 12889, 12890, 12891, 12892, 12893 Extra Details: family 10 hydrolase; glycosyl hydrolase; site-directed mutagenesis;,structure-function; xylanase

Submission Details

ID: 2RQW7XZP4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:44 p.m.

Version: 1

Publication Details
Roberge M;Shareck F;Morosoli R;Kluepfel D;Dupont C,Protein Eng. (1999) Characterization of active-site aromatic residues in xylanase A from Streptomyces lividans. PMID:10235626
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1V0L 2004-08-16 0.98 Xylanase Xyn10A from Streptomyces lividans in complex with xylobio-isofagomine at pH 5.8
1V0K 2004-08-16 1.03 Xylanase Xyn10A from Streptomyces lividans in complex with xylobio-deoxynojirimycin at pH 5.8
1OD8 2003-04-08 1.05 Xylanase Xyn10A from Streptomyces lividans in complex with xylobio-isofagomine lactam
1V0M 2004-08-16 1.07 Xylanase Xyn10a from Streptomyces lividans in complex with xylobio-deoxynojirimycin at pH 7.5
1V0N 2004-08-16 1.1 Xylanase Xyn10a from Streptomyces lividans in complex with xylobio-isofagomine at pH 7.5
1KNM 2002-06-19 1.2 Streptomyces lividans Xylan Binding Domain cbm13 in Complex with Lactose
1KNL 2002-06-19 1.2 Streptomyces lividans Xylan Binding Domain cbm13
1E0W 2001-04-05 1.2 Xylanase 10A from Sreptomyces lividans. native structure at 1.2 angstrom resolution
1E0X 2001-04-05 1.65 XYLANASE 10A FROM SREPTOMYCES LIVIDANS. XYLOBIOSYL-ENZYME INTERMEDIATE AT 1.65 A
1E0V 2001-04-05 1.7 Xylanase 10A from Sreptomyces lividans. cellobiosyl-enzyme intermediate at 1.7 A
1MC9 2002-09-11 1.7 STREPROMYCES LIVIDANS XYLAN BINDING DOMAIN CBM13 IN COMPLEX WITH XYLOPENTAOSE
1XAS 1995-05-31 2.6 CRYSTAL STRUCTURE, AT 2.6 ANGSTROMS RESOLUTION, OF THE STREPTOMYCES LIVIDANS XYLANASE A, A MEMBER OF THE F FAMILY OF BETA-1,4-D-GLYCANSES

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Endo-1,4-beta-xylanase A P26514 XYNA_STRLI