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 ?-d-galactosidase and N-acetyl-?-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 ?-d-galactosidase, ?-d-fucosidase, N-acetyl-?-d-glucosaminidase, and N-acetyl-?-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-?-d-glucosaminide and the highest kcat with 4-methylumbelliferyl-linked ?-d-galactopyranoside. In addition, the purified LacZ domain of MsgA had ?-d-galactosidase and ?-d-fucosidase activities, and the GH20 domain exhibited both N-acetyl-?-d-glucosaminidase and N-acetyl-?-d-galactosaminidase activities. The ?-d-galactosidase and ?-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-?-d-glucosaminidase and N-acetyl-?-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 ?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.

SUBMITTER: Imaki H 

PROVIDER: S-EPMC4135667 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature