Substrate binding to the inactive mutants of Streptomyces sp. N174 chitosanase: indirect evaluation from the thermal unfolding experiments.
Oligosaccharide binding to chitosanase from Streptomyces sp. N174 was indirectly evaluated from thermal unfolding experiments of the protein. Thermal unfolding curves were obtained by fluorescence spectroscopy in the presence of D-glucosamine oligosaccharides ((GlcN)n, n = 3, 4, 5, and 6) using the inactive mutant chitosanase in which the catalytic residue, Glu22, is mutated to glutamine (E22Q), aspartic acid (E22D), or alanine (E22A). The midpoint temperature of the unfolding transition (Tm) of E22Q was found to be 44.4 degrees C at pH 7.0. However, the Tm increased upon the addition of (GlcN), by 1.3 degrees C (n = 3), 2.5 degrees C (n = 4), 5.2 degrees C (n = 5), or 7.6 degrees C (n = 6). No appreciable change in Tm was observed when (GlcNAc)6 was added to E22Q. The effect of (GlcN)n on the thermal stability was examined using the other protein, RNase T1, but the oligosaccharide did not affect Tm of the protein. Thus, we concluded that the stabilization effect of (GlcN)n on the chitosanase results from specific binding of the oligosaccharides to the substrate binding cleft. When E22D or E22A was used instead of E22Q, the increases in Tm induced by (GlcN)6 binding were 2.7 degrees C for E22D and 4.2 degrees C for E22A. In E22D or E22A, interaction with (GlcN)6 seems to be partly disrupted by a conformational distortion in the catalytic cleft.
Honda
Y
Y
Laboratory of Biophysical Chemistry, Faculty of Agriculture, Kinki University, Nara, Japan.
Fukamizo
T
T
Boucher
I
I
Brzezinski
R
R
D000117:Acetylglucosamine / Q000031:analogs & derivatives; D001665:Binding Sites; D005944:Glucosamine / Q000031:analogs & derivatives; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D009844:Oligosaccharides / Q000378:metabolism*; D011485:Protein Binding; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics; D013696:Temperature; D013816:Thermodynamics; D014364:Tryptophan / Q000378:metabolism
D009844:Oligosaccharides; D014364:Tryptophan; D006026:Glycoside Hydrolases; C019868:chitosanase; D005944:Glucosamine; D000117:Acetylglucosamine