title: Truncation of N- and C-terminal regions of Streptococcus mutans dextranase enhances catalytic activity.
abstract: Multiple forms of native and recombinant endo-dextranases (Dexs) of the glycoside hydrolase family (GH) 66 exist. The GH 66 Dex gene from Streptococcus mutans ATCC 25175 (SmDex) was expressed in Escherichia coli. The recombinant full-size (95.4 kDa) SmDex protein was digested to form an 89.8 kDa isoform (SmDex90). The purified SmDex90 was proteolytically degraded to more than seven polypeptides (23-70 kDa) during long storage. The protease-insensitive protein was desirable for the biochemical analysis and utilization of SmDex. GH 66 Dex was predicted to comprise four regions from the N- to C-termini: N-terminal variable region (N-VR), conserved region (CR), glucan-binding site (GBS), and C-terminal variable region (C-VR). Five truncated SmDexs were generated by deleting N-VR, GBS, and/or C-VR. Two truncation-mutant enzymes devoid of C-VR (TM-NCGΔ) or N-VR/C-VR (TM-ΔCGΔ) were catalytically active, thereby indicating that N-VR and C-VR were not essential for the catalytic activity. TM-ΔCGΔ did not accept any further protease-degradation during long storage. TM-NCGΔ and TM-ΔCGΔ enhanced substrate hydrolysis, suggesting that N-VR and C-VR induce hindered substrate binding to the active site.
authors.affiliation: Research Faculty of Agriculture, Hokkaido University, Kita-9 Nishi-9, Kita-ku, Sapporo 060-8589, Japan. u9897854@kribb.re.kr
authors.forename: Young-Min
authors.initials: YM
authors.lastname: Kim
authors.forename: Ryoko
authors.initials: R
authors.lastname: Shimizu
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Nakai
authors.forename: Haruhide
authors.initials: H
authors.lastname: Mori
authors.forename: Masayuki
authors.initials: M
authors.lastname: Okuyama
authors.forename: Min-Sun
authors.initials: MS
authors.lastname: Kang
authors.forename: Zui
authors.initials: Z
authors.lastname: Fujimoto
authors.forename: Kazumi
authors.initials: K
authors.lastname: Funane
authors.forename: Doman
authors.initials: D
authors.lastname: Kim
authors.forename: Atsuo
authors.initials: A
authors.lastname: Kimura
mesh_terms: D000595:Amino Acid Sequence; D055162:Biocatalysis; D001709:Biotechnology / Q000379:methods; D020134:Catalytic Domain; D003910:Dextranase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000145:classification / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017384:Sequence Deletion; D013295:Streptococcus mutans / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; D003910:Dextranase