The Discoidin Domain of<i>Bacillus circulans</i>β-Galactosidase Plays an Essential Role in Repressing Galactooligosaccharide Production

Song, Jingyuan; Imanaka, Hiroyuki; Imamura, Koreyoshi; Minoda, Masashi; Yamaguchi, Shotaro; Nakanishi, Koji

doi:10.1271/bbb.120583

Cited by 11 publications

(18 citation statements)

References 37 publications

(64 reference statements)

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“…between them was 0. From sequence homology, it has been speculated that the structure of BgaD-D consists of four domains [11,15,17]. From the structural analysis, however, we clarified that BgaD-D consists of five domains ( Fig.…”

Section: Resultsmentioning

confidence: 86%

“…The disallowed regions in the Ramachandran plot were observed in Thr98 (A), Trp405 (A), Ser534 (B), Pro646 (A), Thr98 (B), Gly394 (B), Trp405 (B), Ser534 (B) and Glu601 (B). From sequence homology, it has been speculated that the structure of BgaD‐D consists of four domains . From the structural analysis, however, we clarified that BgaD‐D consists of five domains (Fig.…”

Section: Resultsmentioning

confidence: 88%

“…Expression and purification of the recombinant deletion mutant of the β‐galactosidase of B. circulans (BgaD‐D) were carried out with methods described previously . Briefly, E. coli BL21 cells were transformed with the various plasmids prepared above and cultured in Luria–Bertani medium with 50 mg·mL −1 of ampicillin at 37 °C.…”

Section: Methodsmentioning

confidence: 99%

“…Kinetic parameters [Michaelis constant ( K m ) and the maximum reaction rate ( V max )] were determined using the nonlinear least‐squares method . The transglycosylation reaction was assayed using lactose at a final concentration of 5% as substrate in 50 m m sodium acetate buffer (pH 6.0) at 60 °C following the method used previously . The formed oligosaccharides were analyzed by HPLC.…”

Section: Methodsmentioning

confidence: 99%

“…Expression and purification of the recombinant deletion mutant of the b-galactosidase of B. circulans (BgaD-D) were carried out with methods described previously [15,17].…”

Section: Expression and Purification Of Recombinant Proteinsmentioning

confidence: 99%

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Crystal structure of β‐galactosidase from Bacillus circulans ATCC 31382 (BgaD) and the construction of the thermophilic mutants

et al. 2015

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b-Galactosidase (EC 3.2.1.23) from Bacillus circulans ATCC 31382, designated BgaD, exhibits high transglycosylation activity to produce galacto-oligosaccharides. BgaD has been speculated to have a multiple domain architecture including a F5/8-type C domain or a discoidin domain in the C-terminal peptide region from amino acid sequence analysis. Here, we solved the first crystal structure of the C-terminal deletion mutant BgaD-D, consisting of sugar binding, Glyco_hydro, catalytic and bacterial Ig-like domains, at 2.5A. In the asymmetric unit, two molecules of BgaD-D were identified and the value of V M was estimated to be 5.0 A 3 ÁDa À1 . It has been speculated that BgaD-D consists of four domains. From the structural analysis, however, we clarified that BgaD-D consists of five domains. We identified a new domain structure comprised of b-sheets in BgaD. The catalytic domain exhibits a TIM barrel structure with a small pocket suited for accommodating the disaccharides. Detailed structural information for the amino acid residues related to activity and substrate specificity was clarified in the catalytic domain. Furthermore, using the structural information, we successfully constructed some thermostable mutants via protein engineering method. DatabaseCoordinates for the BgaD-D structure have been deposited in the Protein Data Bank under accession code 4YPJ

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Section: Resultsmentioning

confidence: 86%

Section: Resultsmentioning

confidence: 88%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…Expression and purification of the recombinant deletion mutant of the b-galactosidase of B. circulans (BgaD-D) were carried out with methods described previously [15,17].…”

Section: Expression and Purification Of Recombinant Proteinsmentioning

confidence: 99%

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Crystal structure of β‐galactosidase from Bacillus circulans ATCC 31382 (BgaD) and the construction of the thermophilic mutants

et al. 2015

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Mutagenesis of Catalytic Nucleophile of β‐Galactosidase Retains Residual Hydrolytic Activity and Affords a Transgalactosidase

et al. 2021

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Glycosidases that cleave oligosaccharides can also synthesize the glycosidic bond. Site-directed mutagenesis of the catalytic nucleophile commonly abolishes their hydrolytic activity, affording glycosynthases that use glycosyl fluorides as substrates.Here, the synthetic ability of β-galactosidase from Bacillus circulans isoform A (BgaD-A; EC 3.2.1.23, GH2) was investigated by site-directed mutagenesis. The cold-shock expression ensured selective induction and correct folding. Three mutants were constructed at the active-site catalytic nucleophile E532 as putative glycosynthases. However, none of the mutants could process α-galactosyl fluoride as a galactosyl donor. With only negligible hydrolytic activity, two mutants selectively synthesized azido-functionalized N-acetyllactosamine using the pnitrophenyl β-d-galactoside as a galactosyl donor. Thus, they behaved as transglycosidases. This study demonstrates that substitution at the catalytic nucleophile for the assembly of glycosynthases is not unrestrictedly versatile and that the effect of mutagenesis on synthetic abilities depends on the relative orientations of amino acids in the enzyme active site.

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Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2013–2014

Harvey

2018

Mass Spectrometry Reviews

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This review is the eighth update of the original article published in 1999 on the application of Matrix-assisted laser desorption/ionization mass spectrometry (MALDI) mass spectrometry to the analysis of carbohydrates and glycoconjugates and brings coverage of the literature to the end of 2014. Topics covered in the first part of the review include general aspects such as theory of the MALDI process, matrices, derivatization, MALDI imaging, fragmentation, and arrays. The second part of the review is devoted to applications to various structural types such as oligo- and poly- saccharides, glycoproteins, glycolipids, glycosides, and biopharmaceuticals. Much of this material is presented in tabular form. The third part of the review covers medical and industrial applications of the technique, studies of enzyme reactions, and applications to chemical synthesis. © 2018 Wiley Periodicals, Inc. Mass Spec Rev 37:353-491, 2018.

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The Discoidin Domain ofBacillus circulansβ-Galactosidase Plays an Essential Role in Repressing Galactooligosaccharide Production

Cited by 11 publications

References 37 publications

Crystal structure of β‐galactosidase from Bacillus circulans ATCC 31382 (BgaD) and the construction of the thermophilic mutants

Crystal structure of β‐galactosidase from Bacillus circulans ATCC 31382 (BgaD) and the construction of the thermophilic mutants

Mutagenesis of Catalytic Nucleophile of β‐Galactosidase Retains Residual Hydrolytic Activity and Affords a Transgalactosidase

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2013–2014

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