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Molecular cloning and characterization of Bifidobacterium bifidum 1,2-alpha-L-fucosidase (AfcA), a novel inverting glycosidase (glycoside hydrolase family 95).

ABSTRACT: A genomic library of Bifidobacterium bifidum constructed in Escherichia coli was screened for the ability to hydrolyze the alpha-(1-->2) linkage of 2'-fucosyllactose, and a gene encoding 1,2-alpha-l-fucosidase (AfcA) was isolated. The afcA gene was found to comprise 1,959 amino acid residues with a predicted molecular mass of 205 kDa and containing a signal peptide and a membrane anchor at the N and C termini, respectively. A domain responsible for fucosidase activity (the Fuc domain; amino acid residues 577 to 1474) was localized by deletion analysis and then purified as a hexahistidine-tagged protein. The recombinant Fuc domain specifically hydrolyzed the terminal alpha-(1-->2)-fucosidic linkages of various oligosaccharides and a sugar chain of a glycoprotein. The stereochemical course of the hydrolysis of 2'-fucosyllactose was determined to be inversion by using (1)H nuclear magnetic resonance. The primary structure of the Fuc domain exhibited no similarity to those of any glycoside hydrolases (GHs) but showed high similarity to those of several hypothetical proteins in a database. Thus, it was revealed that the AfcA protein constitutes a novel inverting GH family (GH family 95).

SUBMITTER: Katayama T

PROVIDER: S-EPMC451662 | biostudies-literature | 2004 Aug

REPOSITORIES: biostudies-literature

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Molecular cloning and characterization of Bifidobacterium bifidum 1,2-alpha-L-fucosidase (AfcA), a novel inverting glycosidase (glycoside hydrolase family 95).

Katayama Takane T Sakuma Akiko A Kimura Takatoshi T Makimura Yutaka Y Hiratake Jun J Sakata Kanzo K Yamanoi Takashi T Kumagai Hidehiko H Yamamoto Kenji K

Journal of bacteriology 20040801 15

A genomic library of Bifidobacterium bifidum constructed in Escherichia coli was screened for the ability to hydrolyze the alpha-(1-->2) linkage of 2'-fucosyllactose, and a gene encoding 1,2-alpha-l-fucosidase (AfcA) was isolated. The afcA gene was found to comprise 1,959 amino acid residues with a predicted molecular mass of 205 kDa and containing a signal peptide and a membrane anchor at the N and C termini, respectively. A domain responsible for fucosidase activity (the Fuc domain; amino acid ...[more]

PMID: 15262925

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Project description:Uronic acids are commonly found in marine polysaccharides and increase structural complexity and intrinsic recalcitrance to enzymatic attack. Glycoside hydrolase family 2 (GH2) includes proteins that target sugar conjugates with hexuronates and are involved in the catabolism and cycling of marine polysaccharides. Here, we report a novel GH2, AqGalA from a marine alga-associated Bacteroidetes organism with broad substrate specificity. Biochemical analyses revealed that AqGalA exhibits hydrolyzing activities against β-galacturonide, β-glucuronide, and β-galactopyranoside via retaining mechanisms. We solved the AqGalA crystal structure in complex with galacturonic acid (GalA) and determined (via mutagenesis) that charge characteristics at uronate-binding subsites controlled substrate selectivity for uronide hydrolysis. Additionally, conformational flexibility of the AqGalA active-site pocket was proposed as a key component for broad substrate enzyme selectivity. Our AqGalA structural and functional data augment the current understanding of substrate recognition of GH2 enzymes and provide key insights into the bacterial use of uronic acid-containing polysaccharides. IMPORTANCE The decomposition of algal glycans driven by marine bacterial communities represents one of the largest heterotrophic transformations of organic matter fueling marine food webs and global carbon cycling. However, our knowledge on carbohydrate cycling is limited due to structural complexity of marine polysaccharides and the complicated enzymatic machinery of marine microbes. To degrade algal glycan, marine bacteria such as members of the Bacteroidetes produce a complex repertoire of carbohydrate-active enzymes (CAZymes) matching the structural specificities of the different carbohydrates. In this study, we investigated an extracellular GH2 β-glycosidase, AqGalA from a marine Bacteroidetes organism, to identify the key components responsible for glycuronide recognition and hydrolysis. The broad substrate specificity of AqGalA against glycosides with diverse stereochemical substitutions indicates its potential in processing complex marine polysaccharides. Our findings promote a better understanding of microbially driven mechanisms of marine carbohydrate cycling.

Dataset Information

Molecular cloning and characterization of Bifidobacterium bifidum 1,2-alpha-L-fucosidase (AfcA), a novel inverting glycosidase (glycoside hydrolase family 95).

Publications

Molecular cloning and characterization of Bifidobacterium bifidum 1,2-alpha-L-fucosidase (AfcA), a novel inverting glycosidase (glycoside hydrolase family 95).

Similar Datasets

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