title: The discoidin domain of Bacillus circulans β-galactosidase plays an essential role in repressing galactooligosaccharide production.
abstract: The recently cloned β-galactosidase from Bacillus circulans ATCC 31382, designated BgaD, contains a multiple domain architecture including a F5/8 type C domain or a discoidin (DS) domain in the C-terminal peptide region. Here we report that the DS domain plays an essential role in repressing the production of galactooligosaccharides (GOSs). We prepared deletion mutants and point-mutated forms of rBgaD-A (deletion of the BgaD signal peptide) to compare their reaction behaviors. The yields of GOSs for all of the point-mutated forms as well as the deletion mutants of rBgaD-As increased as compared to rBgaD-A. In particular, W1540A mutant BgaD-A (rBgaD-A_W1540A) produced much more GOSs than rBgaD-A. Surface plasmon resonance experiments indicated that both the wild-type and the W1540A mutant DS domains showed high affinity for galactosyllactose. rBgaD-A, which has a wild-type DS domain, showed high hydrolytic activity toward galactosyllactose, while the hydrolytic activities of rBgaD-D, without a DS domain, and rBgaD-A_W1540A, with a mutant DS domain were extremely low. The findings obtained in this study indicate that the wild-type DS domain of rBgaD-A has a function that aids galactosyllactose molecules to be properly oriented within the active site, so that they can be hydrolyzed efficiently to produce galactose/glucose by inhibiting the accumulation of GOSs.
authors.affiliation: Graduate School of Natural Science and Technology, Okayama University, Japan.
authors.forename: Jingyuan
authors.initials: J
authors.lastname: Song
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Imanaka
authors.forename: Koreyoshi
authors.initials: K
authors.lastname: Imamura
authors.forename: Masashi
authors.initials: M
authors.lastname: Minoda
authors.forename: Shotaro
authors.initials: S
authors.lastname: Yamaguchi
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Nakanishi
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000070821:Discoidins; D004926:Escherichia coli / Q000235:genetics; D005690:Galactose / Q000096:biosynthesis; D005697:Galactosides / Q000096:biosynthesis*; D007785:Lactose / Q000096:biosynthesis; D037102:Lectins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D001616:beta-Galactosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D000070821:Discoidins; D005697:Galactosides; D037102:Lectins; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D001616:beta-Galactosidase; D007785:Lactose; D005690:Galactose