Functional analysis of conserved aromatic amino acids in the discoidin domain of Paenibacillus beta-1,3-glucanase.
The 190-kDa Paenibacillus beta-1,3-glucanase (LamA) contains a catalytic module of the glycoside hydrolase family 16 (GH16) and several auxiliary domains. Of these, a discoidin domain (DS domain), present in both eukaryotic and prokaryotic proteins with a wide variety of functions, exists at the carboxyl-terminus. To better understand the bacterial DS domain in terms of its structure and function, this domain alone was expressed in Escherichia coli and characterized. The results indicate that the DS domain binds various polysaccharides and enhances the biological activity of the GH16 module on composite substrates. We also investigated the importance of several conserved aromatic residues in the domain's stability and substrate-binding affinity. Both were affected by mutations of these residues; however, the effect on protein stability was more notable. In particular, the forces contributed by a sandwiched triad (W1688, R1756, and W1729) were critical for the presumable beta-sandwich fold.
Cheng
Yueh-Mei
YM
Graduate Institute of Biotechnology, National Chung Hsing University, 250 Kuo-Kuang Rd, Taichung, 40227, Taiwan. q4351@yahoo.com.tw
Hsieh
Feng-Chia
FC
Meng
Menghsiao
M
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D024322:Amino Acids, Aromatic / Q000737:chemistry*; D001665:Binding Sites; D000070821:Discoidins; D043326:Glucan 1,3-beta-Glucosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D037102:Lectins / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D056507:Paenibacillus / Q000201:enzymology*; D011134:Polysaccharides / Q000378:metabolism; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
D024322:Amino Acids, Aromatic; D000070821:Discoidins; D037102:Lectins; D011134:Polysaccharides; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D043326:Glucan 1,3-beta-Glucosidase