title: Identification and characterization of a novel secreted glycosidase with multiple glycosidase activities in Streptococcus intermedius.
abstract: Streptococcus intermedius is a known human pathogen and belongs to the anginosus group (S. anginosus, S. intermedius, and S. constellatus) of streptococci (AGS). We found a large open reading frame (6,708 bp) in the lac operon, and bioinformatic analysis suggested that this gene encodes a novel glycosidase that can exhibit β-d-galactosidase and N-acetyl-β-d-hexosaminidase activities. We, therefore, named this protein "multisubstrate glycosidase A" (MsgA). To test whether MsgA has these glycosidase activities, the msgA gene was disrupted in S. intermedius. The msgA-deficient mutant no longer showed cell- and supernatant-associated β-d-galactosidase, β-d-fucosidase, N-acetyl-β-d-glucosaminidase, and N-acetyl-β-d-galactosaminidase activities, and all phenotypes were complemented in trans with a recombinant plasmid carrying msgA. Purified MsgA had all four of these glycosidase activities and exhibited the lowest Km with 4-methylumbelliferyl-linked N-acetyl-β-d-glucosaminide and the highest kcat with 4-methylumbelliferyl-linked β-d-galactopyranoside. In addition, the purified LacZ domain of MsgA had β-d-galactosidase and β-d-fucosidase activities, and the GH20 domain exhibited both N-acetyl-β-d-glucosaminidase and N-acetyl-β-d-galactosaminidase activities. The β-d-galactosidase and β-d-fucosidase activities of MsgA are thermolabile, and the optimal temperature of the reaction was 40°C, whereas almost all enzymatic activities disappeared at 49°C. The optimal temperatures for the N-acetyl-β-d-glucosaminidase and N-acetyl-β-d-galactosaminidase activities were 58 and 55°C, respectively. The requirement of sialidase treatment to remove sialic acid residues of the glycan branch end for glycan degradation by MsgA on human α1-antitrypsin indicates that MsgA has exoglycosidase activities. MsgA and sialidase might have an important function in the production and utilization of monosaccharides from oligosaccharides, such as glycans for survival in a normal habitat and for pathogenicity of S. intermedius.
authors.affiliation: Department of Biological Science and Technology, Institute of Technology and Science, The University of Tokushima Graduate School, Tokushima, Japan.
authors.forename: Hidenori
authors.initials: H
authors.lastname: Imaki
authors.affiliation: Department of Biological Science and Technology, Institute of Technology and Science, The University of Tokushima Graduate School, Tokushima, Japan Department of Resource Circulation Engineering, Center for Frontier Research of Engineering, The University of Tokushima Graduate School, Tokushima, Japan.
authors.forename: Toshifumi
authors.initials: T
authors.lastname: Tomoyasu
authors.affiliation: Department of Biological Science and Technology, Institute of Technology and Science, The University of Tokushima Graduate School, Tokushima, Japan.
authors.forename: Naoki
authors.initials: N
authors.lastname: Yamamoto
authors.affiliation: Department of Biological Science and Technology, Institute of Technology and Science, The University of Tokushima Graduate School, Tokushima, Japan.
authors.forename: Chiharu
authors.initials: C
authors.lastname: Taue
authors.affiliation: Department of Biological Science and Technology, Institute of Technology and Science, The University of Tokushima Graduate School, Tokushima, Japan.
authors.forename: Sachiko
authors.initials: S
authors.lastname: Masuda
authors.affiliation: Department of Oral Microbiology, School of Dental Medicine, Tsurumi University, Yokohama, Japan.
authors.forename: Ayuko
authors.initials: A
authors.lastname: Takao
authors.affiliation: Department of Oral Microbiology, School of Dental Medicine, Tsurumi University, Yokohama, Japan.
authors.forename: Nobuko
authors.initials: N
authors.lastname: Maeda
authors.affiliation: Department of Biological Science and Technology, Institute of Technology and Science, The University of Tokushima Graduate School, Tokushima, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Tabata
authors.affiliation: Department of Clinical and Diagnostic Oral Sciences, Institute of Dentistry, Bart's and The London School of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Whiley
authors.affiliation: Department of Biological Science and Technology, Institute of Technology and Science, The University of Tokushima Graduate School, Tokushima, Japan nagamune@bio.tokushima-u.ac.jp.
authors.forename: Hideaki
authors.initials: H
authors.lastname: Nagamune
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002876:Chromosomes, Bacterial / Q000235:genetics*; D019295:Computational Biology; D055786:Gene Knockout Techniques; D017930:Genes, Reporter; D005816:Genetic Complementation Test; D006026:Glycoside Hydrolases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D011134:Polysaccharides / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins; D017384:Sequence Deletion; D034367:Streptococcus intermedius / Q000201:enzymology* / Q000235:genetics; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D011134:Polysaccharides; D011993:Recombinant Fusion Proteins; D006026:Glycoside Hydrolases