title: Functional dissection of the N-terminal sequence of Clostridium sp. G0005 glucoamylase: identification of components critical for folding the catalytic domain and for constructing the active site structure.
abstract: Clostridium sp. G0005 glucoamylase (CGA) is composed of a β-sandwich domain (BD), a linker, and a catalytic domain (CD). In the present study, CGA was expressed in Escherichia coli as inclusion bodies when the N-terminal region (39 amino acid residues) of the BD was truncated. To further elucidate the role of the N-terminal region of the BD, we constructed N-terminally truncated proteins (Δ19, Δ24, Δ29, and Δ34) and assessed their solubility and activity. Although all evaluated proteins were soluble, their hydrolytic activities toward maltotriose as a substrate varied: Δ19 and Δ24 were almost as active as CGA, but the activity of Δ29 was substantially lower, and Δ34 exhibited little hydrolytic activity. Subsequent truncation analysis of the N-terminal region sequence between residues 25 and 28 revealed that truncation of less than 26 residues did not affect CGA activity, whereas truncation of 26 or more residues resulted in a substantial loss of activity. Based on further site-directed mutagenesis and N-terminal sequence analysis, we concluded that the 26XaaXaaTrp28 sequence of CGA is important in exhibiting CGA activity. These results suggest that the N-terminal region of the BD in bacterial GAs may function not only in folding the protein into the correct structure but also in constructing a competent active site for catalyzing the hydrolytic reaction.
authors.affiliation: Department of Chemistry and Life Science, Kogakuin University, 2,665-1 Nakano-cho, Hachioji, Tokyo, 192-0015, Japan. bt11532@ns.kogakuin.ac.jp.
authors.forename: Masayoshi
authors.identifier: http://orcid.org/0000-0001-6724-9669
authors.initials: M
authors.lastname: Sakaguchi
authors.affiliation: Department of Chemistry and Life Science, Kogakuin University, 2,665-1 Nakano-cho, Hachioji, Tokyo, 192-0015, Japan.
authors.forename: Yudai
authors.initials: Y
authors.lastname: Matsushima
authors.affiliation: Department of Chemistry and Life Science, Kogakuin University, 2,665-1 Nakano-cho, Hachioji, Tokyo, 192-0015, Japan.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Nagamine
authors.affiliation: Department of Chemistry and Life Science, Kogakuin University, 2,665-1 Nakano-cho, Hachioji, Tokyo, 192-0015, Japan.
authors.forename: Tomoki
authors.initials: T
authors.lastname: Matsuhashi
authors.affiliation: Department of Chemistry and Life Science, Kogakuin University, 2,665-1 Nakano-cho, Hachioji, Tokyo, 192-0015, Japan.
authors.forename: Shotaro
authors.initials: S
authors.lastname: Honda
authors.affiliation: Department of Chemistry and Life Science, Kogakuin University, 2,665-1 Nakano-cho, Hachioji, Tokyo, 192-0015, Japan.
authors.forename: Shoi
authors.initials: S
authors.lastname: Okuda
authors.affiliation: Department of Chemistry and Life Science, Kogakuin University, 2,665-1 Nakano-cho, Hachioji, Tokyo, 192-0015, Japan.
authors.forename: Misa
authors.initials: M
authors.lastname: Ohno
authors.affiliation: Department of Chemistry and Life Science, Kogakuin University, 2,665-1 Nakano-cho, Hachioji, Tokyo, 192-0015, Japan.
authors.forename: Yasusato
authors.initials: Y
authors.lastname: Sugahara
authors.affiliation: Department of Chemistry and Life Science, Kogakuin University, 2,665-1 Nakano-cho, Hachioji, Tokyo, 192-0015, Japan.
authors.forename: Yongchol
authors.initials: Y
authors.lastname: Shin
authors.affiliation: Department of Chemistry and Life Science, Kogakuin University, 2,665-1 Nakano-cho, Hachioji, Tokyo, 192-0015, Japan.
authors.forename: Fumitaka
authors.initials: F
authors.lastname: Oyama
authors.affiliation: Department of Chemistry and Life Science, Kogakuin University, 2,665-1 Nakano-cho, Hachioji, Tokyo, 192-0015, Japan.|Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kami-kitazawa, Setagaya-ku, Tokyo, 156-8506, Japan.
authors.forename: Masao
authors.initials: M
authors.lastname: Kawakita
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D003001:Cloning, Molecular; D003013:Clostridium / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005087:Glucan 1,4-alpha-Glucosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D002479:Inclusion Bodies / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014312:Trisaccharides / Q000737:chemistry* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D014312:Trisaccharides; C008317:maltotriose; D005087:Glucan 1,4-alpha-Glucosidase
keywords: GH15 family enzymes; Glucoamylase; Inclusion body; Internal chaperone-like function; N-terminal region loop