Identification and Characterization of a Novel Secreted Glycosidase with Multiple Glycosidase Activities in Streptococcus intermedius

Abstract

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 K m with 4-methylumbelliferyl-linked N -acetyl-β- d -glucosaminide and the highest k cat 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 α 1 -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 .