Three N-terminal domains of beta-1,3-glucanase A1 are involved in binding to insoluble beta-1,3-glucan.
Limited proteolysis of beta-1,3-glucanase A1 by three different proteases, trypsin, chymotrypsin, and papain, gave three major active fragments. The sizes of the three major fragments generated by each protease treatment were identical to those of beta-1,3-glucanase A2, A3, and A4 detected in both the culture supernatant of Bacillus circulans WL-12 and the periplasmic space of Escherichia coli carrying a cloned glcA gene. These results indicate a four-domain structure for the enzyme. At the N terminus of the glucanase, duplicated segments of approximately 100 amino acids were observed. N-terminal amino acid sequence analysis revealed that the active fragments with sizes corresponding to those of A2 and A3 lack the first segment (domain) and both duplicated segments (domains), respectively. The fragment corresponding to A4 lacks both duplicated segments and the following ca. 120-amino-acid region. By losing the first, second, and third (corresponding to the segment of 120 amino acids) domains, beta-1,3-glucanase progressively lost the ability to bind to pachyman, beta-1,3-glucan. An active fragment which did not have the three N-terminal domains did not show significant binding to pachyman. Thus, all three N-terminal domains contribute to binding to beta-1,3-glucan, and the presence of three domains confers the highest binding activity on the glucanase. The loss of these binding domains remarkably decreased pachyman-hydrolyzing activity, indicating that the binding activity is essential for the efficient hydrolysis of insoluble beta-1,3-glucan.
Watanabe
T
T
Department of Agricultural Chemistry, Faculty of Agriculture, Niigata University, Japan.
Kasahara
N
N
Aida
K
K
Tanaka
H
H
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites; D002918:Chymotrypsin / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D043326:Glucan 1,3-beta-Glucosidase; D005936:Glucans / Q000378:metabolism*; D008969:Molecular Sequence Data; D010206:Papain / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism; D011487:Protein Conformation; D012689:Sequence Homology, Nucleic Acid; D014357:Trypsin / Q000378:metabolism; D047071:beta-Glucans; D001617:beta-Glucosidase / Q000737:chemistry* / Q000235:genetics
D005936:Glucans; D010446:Peptide Fragments; D047071:beta-Glucans; C014731:pachyman; C033363:beta-1,3-glucan; D001617:beta-Glucosidase; D043326:Glucan 1,3-beta-Glucosidase; D010447:Peptide Hydrolases; D002918:Chymotrypsin; D014357:Trypsin; D010206:Papain