Crystallization and preliminary X-ray crystallographic analysis of α-glucosidase HaG from<i>Halomonas</i>sp. strain H11

Shen, Xing; Saburi, Wataru; Gai, Zuoqi; Komoda, Keisuke; Yu, Jian; Ojima-Kato, Teruyo; Kido, Yusuke; Matsui, Hirokazu; Mori, Haruhide; Yao, Min

doi:10.1107/s2053230x14001940

Cited by 4 publications

(9 citation statements)

References 24 publications

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“…Wild-type HaG was expressed in Escherichia coli BL21(DE3) cells harbouring the recombinant plasmid pFLAG-CTS and purified using a DEAE-650M anionexchange column (Tosoh, Tokyo, Japan) as described elsewhere (Shen et al, 2014). Expression plasmids for two activesite mutants, D202N and E271Q, were created by site-directed mutagenesis with a Primestar Mutagenesis Basal Kit (Takara Bio, Otsu, Japan).…”

Section: Plasmid Construction Expression and Purification Of Recombimentioning

confidence: 99%

“…Expression plasmids for two activesite mutants, D202N and E271Q, were created by site-directed mutagenesis with a Primestar Mutagenesis Basal Kit (Takara Bio, Otsu, Japan). The mutant enzymes were prepared using the same protocol as for the wild-type HaG (Shen et al, 2014).…”

Section: Plasmid Construction Expression and Purification Of Recombimentioning

confidence: 99%

“…Crystals of wild-type HaG were grown in 0.1 M HEPES-NaOH buffer pH 7.0 containing 0.02 M magnesium chloride and 20% polyacrylic acid (PAA) 5100 sodium salt as described previously (Shen et al, 2014).…”

Section: Crystallization and Data Collectionmentioning

confidence: 99%

“…The structure of HaG was determined by the molecularreplacement (MR) method with Phaser (McCoy et al, 2007), using the monomer structure of the isomaltulose synthase PalI from Klebsiella sp. LX3 (PDB entry 1m53; 36% identity to HaG; Zhang et al, 2003) as a search model (Shen et al, 2014).…”

Section: Structure Determination and Refinementmentioning

confidence: 99%

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Structural analysis of the α-glucosidase HaG provides new insights into substrate specificity and catalytic mechanism

Shen

Saburi

Gai

et al. 2015

Acta Crystallogr D Biol Crystallogr

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α-Glucosidases, which catalyze the hydrolysis of the α-glucosidic linkage at the nonreducing end of the substrate, are important for the metabolism of α-glucosides. Halomonas sp. H11 α-glucosidase (HaG), belonging to glycoside hydrolase family 13 (GH13), only has high hydrolytic activity towards the α-(1 → 4)-linked disaccharide maltose among naturally occurring substrates. Although several three-dimensional structures of GH13 members have been solved, the disaccharide specificity and α-(1 → 4) recognition mechanism of α-glucosidase are unclear owing to a lack of corresponding substrate-bound structures. In this study, four crystal structures of HaG were solved: the apo form, the glucosyl-enzyme intermediate complex, the E271Q mutant in complex with its natural substrate maltose and a complex of the D202N mutant with D-glucose and glycerol. These structures explicitly provide insights into the substrate specificity and catalytic mechanism of HaG. A peculiar long β → α loop 4 which exists in α-glucosidase is responsible for the strict recognition of disaccharides owing to steric hindrance. Two residues, Thr203 and Phe297, assisted with Gly228, were found to determine the glycosidic linkage specificity of the substrate at subsite +1. Furthermore, an explanation of the α-glucosidase reaction mechanism is proposed based on the glucosyl-enzyme intermediate structure.

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Section: Plasmid Construction Expression and Purification Of Recombimentioning

confidence: 99%

Section: Plasmid Construction Expression and Purification Of Recombimentioning

confidence: 99%

Section: Crystallization and Data Collectionmentioning

confidence: 99%

Section: Structure Determination and Refinementmentioning

confidence: 99%

See 2 more Smart Citations

Structural analysis of the α-glucosidase HaG provides new insights into substrate specificity and catalytic mechanism

Shen

Saburi

Gai

et al. 2015

Acta Crystallogr D Biol Crystallogr

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“…strain HTA-462 (Shirai et al 2008) and Halomonas sp. strain H11 (Shen et al 2014) are included in this family. By contrast, family GH31 includes important enzymes such as the human lysosomal α-glucosidase (whose deficiency results in the Pompe's disease; Hermans et al 1991;Raben et al 2002) or the sucrose-isomaltase (target of inhibition of some anti-diabetes drugs; Mohan et al 2014), much less data concerning this protein family has been clarified.…”

Section: Introductionmentioning

confidence: 99%

Molecular characterization and heterologous expression of a Xanthophyllomyces dendrorhous α-glucosidase with potential for prebiotics production

Gutiérrez-Alonso

Gimeno-Pérez

Ramirez-Escudero

et al. 2015

Appl Microbiol Biotechnol

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Basidiomycetous yeast Xanthophyllomyces dendrorhous expresses an α-glucosidase with strong transglycosylation activity producing prebiotic sugars such as panose and an unusual tetrasaccharides mixture including α-(1-6) bonds as major products, which makes it of biotechnological interest. Initial analysis pointed to a homodimeric protein of 60 kDa subunit as responsible for this activity. In this study, the gene Xd-AlphaGlu was characterized. The 4131-bp-long gene is interrupted by 13 short introns and encodes a protein of 990 amino acids (Xd-AlphaGlu). The N-terminal sequence of the previously detected 60 kDa protein resides in this larger protein at residues 583-602. Functionality of the gene was proved in Saccharomyces cerevisiae, which produced a protein of about 130 kDa containing Xd-AlphaGlu sequences. All properties of the heterologously expressed protein, including thermal and pH profiles, activity on different substrates, and ability to produce prebiotic sugars were similar to that of the α-glucosidase produced in X. dendrorhous. No activity was detected in S. cerevisiae containing exclusively the 1256-bp from gene Xd-AlphaGlu that would encode synthesis of the 60 kDa protein previously detected. Data were compatible with an active monomeric α-glucosidase of 990 amino acids and an inactive hydrolysis product of 60 kDa. Protein Xd-AlphaGlu contained most of the elements characteristic of α-glucosidases included in the glycoside hydrolases family GH31 and its structural model based on the homologous human maltase-glucoamylase was obtained. Remarkably, the Xd-AlphaGlu C-terminal domain presents an unusually long 115-residue insertion that could be involved in this enzyme's activity against long-size substrates such as maltoheptaose and soluble starch.

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Enzymatic and selective production of alkyl α-d-glucopyranosides by the α-glucosyl transfer enzyme derived from Xanthomonas campestris WU-9701

Cao,

Watanabe,

Ishii

et al. 2023

Journal of Bioscience and Bioengineering

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Crystallization and preliminary X-ray crystallographic analysis of α-glucosidase HaG fromHalomonassp. strain H11

Cited by 4 publications

References 24 publications

Structural analysis of the α-glucosidase HaG provides new insights into substrate specificity and catalytic mechanism

Structural analysis of the α-glucosidase HaG provides new insights into substrate specificity and catalytic mechanism

Molecular characterization and heterologous expression of a Xanthophyllomyces dendrorhous α-glucosidase with potential for prebiotics production

Enzymatic and selective production of alkyl α-d-glucopyranosides by the α-glucosyl transfer enzyme derived from Xanthomonas campestris WU-9701

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