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Streptococcus pneumoniae endohexosaminidase D, structural and mechanistic insight into substrate-assisted catalysis in family 85 glycoside hydrolases.

ABSTRACT: Endo-beta-d-glucosaminidases from family 85 of glycoside hydrolases (GH85 endohexosaminidases) act to cleave the glycosidic linkage between the two N-acetylglucosamine units that make up the chitobiose core of N-glycans. Endohexosaminidase D (Endo-D), produced by Streptococcus pneumoniae, is believed to contribute to the virulence of this organism by playing a role in the deglycosylation of IgG antibodies. Endohexosaminidases have received significant attention for this reason and, moreover, because they are powerful tools for chemoenzymatic synthesis of proteins having defined glycoforms. Here we describe mechanistic and structural studies of the catalytic domain (SpGH85) of Endo-D that provide compelling support for GH85 enzymes using a catalytic mechanism involving substrate-assisted catalysis. Furthermore, the structure of SpGH85 in complex with the mechanism-based competitive inhibitor NAG-thiazoline (K(d) = 28 microm) provides a coherent rationale for previous mutagenesis studies of Endo-D and other related GH85 enzymes. We also find GH85, GH56, and GH18 enzymes have a similar configuration of catalytic residues. Notably, GH85 enzymes have an asparagine in place of the aspartate residue found in these other families of glycosidases. We propose that this residue, as the imidic acid tautomer, acts analogously to the key catalytic aspartate of GH56 and GH18 enzymes. This topographically conserved arrangement of the asparagine residue and a conserved glutamic acid, coupled with previous kinetic studies, suggests these enzymes may use an unusual proton shuttle to coordinate effective general acid and base catalysis to aid cleavage of the glycosidic bond. These results collectively provide a blueprint that may be used to facilitate protein engineering of these enzymes to improve their function as biocatalysts for synthesizing glycoproteins having defined glycoforms and also may serve as a guide for generating inhibitors of GH85 enzymes.

SUBMITTER: Abbott DW

PROVIDER: S-EPMC2670171 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

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Streptococcus pneumoniae endohexosaminidase D, structural and mechanistic insight into substrate-assisted catalysis in family 85 glycoside hydrolases.

Abbott D Wade DW Macauley Matthew S MS Vocadlo David J DJ Boraston Alisdair B AB

The Journal of biological chemistry 20090130 17

Endo-beta-d-glucosaminidases from family 85 of glycoside hydrolases (GH85 endohexosaminidases) act to cleave the glycosidic linkage between the two N-acetylglucosamine units that make up the chitobiose core of N-glycans. Endohexosaminidase D (Endo-D), produced by Streptococcus pneumoniae, is believed to contribute to the virulence of this organism by playing a role in the deglycosylation of IgG antibodies. Endohexosaminidases have received significant attention for this reason and, moreover, bec ...[more]

PMID: 19181667

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Project description:UnlabelledThe growth of the oral commensal Streptococcus gordonii in saliva may depend on a number of glycoside hydrolases (GHs), including three cell wall-anchored proteins that are homologs of pneumococcal β-galactosidase (BgaA), β-N-acetylglucosaminidase (StrH), and endo-β-N-acetylglucosaminidase D (EndoD). In the present study, we introduced unmarked in-frame deletions into the corresponding genes of S. gordonii DL1, verified the presence (or absence) of the encoded proteins on the resulting mutant strains, and compared these strains with wild-type strain DL1 for growth and glycan foraging in saliva. The overnight growth of wild-type DL1 was reduced 3- to 10-fold by the deletion of any one or two genes and approximately 20-fold by the deletion of all three genes. The only notable change in the salivary proteome associated with this reduction of growth was a downward shift in the apparent molecular masses of basic proline-rich glycoproteins (PRG), which was accompanied by the loss of lectin binding sites for galactose-specific Erythrina cristagalli agglutinin (ECA) and mannose-specific Galanthus nivalis agglutinin (GNA). The binding of ECA to PRG was also abolished in saliva cultures of mutants that expressed cell surface BgaA alone or together with either StrH or EndoD. However, the subsequent loss of GNA binding was seen only in saliva cocultures of different mutants that together expressed all three cell surface GHs. The findings indicate that the growth of S. gordonii DL1 in saliva depends to a significant extent on the sequential actions of first BgaA and then StrH and EndoD on N-linked glycans of PRG.ImportanceThe ability of oral bacteria to grow on salivary glycoproteins is critical for dental plaque biofilm development. Little is known, however, about how specific salivary components are attacked and utilized by different members of the biofilm community, such as Streptococcus gordonii. Streptococcus gordonii DL1 has three cell wall-anchored glycoside hydrolases that are predicted to act on host glycans. In the present study, we introduced unmarked in-frame deletions in the corresponding genes, verified the presence (or absence) of encoded proteins on the resulting mutant strains, and compared these strains with wild-type DL1 for growth and glycan foraging in saliva. The results indicate that the growth of S. gordonii DL1 depends to a significant extent on sequential action of these cell surface GHs on N-linked glycans of basic proline-rich salivary glycoproteins, which appears to be an essential first step in salivary glycan foraging.

Dataset Information

Streptococcus pneumoniae endohexosaminidase D, structural and mechanistic insight into substrate-assisted catalysis in family 85 glycoside hydrolases.

Publications

Streptococcus pneumoniae endohexosaminidase D, structural and mechanistic insight into substrate-assisted catalysis in family 85 glycoside hydrolases.

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