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The highly conserved domain of unknown function 1792 has a distinct glycosyltransferase fold.

Zhang H, Zhu F, Yang T, Ding L, Zhou M, Li J, Haslam SM, Dell A, Erlandsen H, Wu H - Nat Commun (2014)

Bottom Line: Structural studies, however, have only revealed two distinct glycosyltransferase (GT) folds, GT-A and GT-B.Biochemical studies reveal that the domain is a glucosyltransferase, and it catalyses the transfer of glucose to the branch point of the hexasaccharide O-linked to the serine-rich repeat of the bacterial adhesin, Fap1 of Streptococcus parasanguinis.Thus, DUF1792 represents a new family of glycosyltransferases; therefore, we designate it as a GT-D glycosyltransferase fold.

View Article: PubMed Central - PubMed

Affiliation: Departments of Pediatric Dentistry, Microbiology, Schools of Dentistry and Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.

ABSTRACT
More than 33,000 glycosyltransferases have been identified. Structural studies, however, have only revealed two distinct glycosyltransferase (GT) folds, GT-A and GT-B. Here we report a 1.34-Å resolution X-ray crystallographic structure of a previously uncharacterized 'domain of unknown function' 1792 (DUF1792) and show that the domain adopts a new fold and is required for glycosylation of a family of serine-rich repeat streptococcal adhesins. Biochemical studies reveal that the domain is a glucosyltransferase, and it catalyses the transfer of glucose to the branch point of the hexasaccharide O-linked to the serine-rich repeat of the bacterial adhesin, Fap1 of Streptococcus parasanguinis. DUF1792 homologues from both Gram-positive and Gram-negative bacteria also exhibit the activity. Thus, DUF1792 represents a new family of glycosyltransferases; therefore, we designate it as a GT-D glycosyltransferase fold. As the domain is highly conserved in bacteria and not found in eukaryotes, it can be explored as a new antibacterial target.

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The glycosyltransferase activity and DXE motif of DUF1792 are conserved(a) DUF1792 is functionally conserved. DUF1792 homologs from a variety of bacterial species were cloned into vector pGEx-6p-1 and co-transformed in E. coli carrying rFap1 and Gtf1/2, 3 to determine the ability of DUF1792 to catalyze the transfer of additional sugar residues to Gtf1/2, 3 modified rFap1. Cell lysates from rFap1 (1), Gtf1/2, 3 modified rFap1 (2) and Gtf1/2, 3 modified rFap1coexpressed with dGT1 of S. parasanguinis (3), or with DUF1792 from S. parasanguinis (4), S. agalactiaeCOH1 (5); S. sanguinis SK36 (6), S. agalactiae 2603 V/R (7), S. pneumoniae TIGR4 (8) and F. nucleatum F0401 (9) were subjected to western blotting analysis with Fap1 specific antibody E42. All DUF1792 homologs retarded the migration of rFap1, suggesting they promoted the transfer of additional sugar residues to Gtf1/2, 3 modified Fap1.(b) Contribution of DUF1792 to glycosylation of Fap1. Wild type dGT1, N-terminal dGT1-DUF1792, C-terminal dGT1 constructs were used to complement the dGT1mutant in S. parasanguinis. Cell lysates from wild type S. parasanguinis (1); Fap1 mutant (2); dGT1 mutant (3); the dGT1 mutant complemented with the dGT1 full-length gene (4), DUF1792 (5); and C-terminal dGT1 (6) were subjected to western blotting analysis with Fap1-peptide specific mAbE42 (top) and anti-DNAK antibody (bottom)as a sample loading control. Complementation of DUF1792 produces an intermediate of Fap1, while the complementation with the C-terminus alone has the same phenotype as dGT1 mutant.(c)The DXE motif and UDP binding sites are highly conserved in bacteria. DUF1792 homologs were identified from a group of streptococci, Lactobacillus lactis and several Gram-negative bacteria. The regions flanking the DGE motif, and UDP binding sites were compared. The invariant amino acid residues highlighted in red and consensus sequence was deduced.
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Figure 3: The glycosyltransferase activity and DXE motif of DUF1792 are conserved(a) DUF1792 is functionally conserved. DUF1792 homologs from a variety of bacterial species were cloned into vector pGEx-6p-1 and co-transformed in E. coli carrying rFap1 and Gtf1/2, 3 to determine the ability of DUF1792 to catalyze the transfer of additional sugar residues to Gtf1/2, 3 modified rFap1. Cell lysates from rFap1 (1), Gtf1/2, 3 modified rFap1 (2) and Gtf1/2, 3 modified rFap1coexpressed with dGT1 of S. parasanguinis (3), or with DUF1792 from S. parasanguinis (4), S. agalactiaeCOH1 (5); S. sanguinis SK36 (6), S. agalactiae 2603 V/R (7), S. pneumoniae TIGR4 (8) and F. nucleatum F0401 (9) were subjected to western blotting analysis with Fap1 specific antibody E42. All DUF1792 homologs retarded the migration of rFap1, suggesting they promoted the transfer of additional sugar residues to Gtf1/2, 3 modified Fap1.(b) Contribution of DUF1792 to glycosylation of Fap1. Wild type dGT1, N-terminal dGT1-DUF1792, C-terminal dGT1 constructs were used to complement the dGT1mutant in S. parasanguinis. Cell lysates from wild type S. parasanguinis (1); Fap1 mutant (2); dGT1 mutant (3); the dGT1 mutant complemented with the dGT1 full-length gene (4), DUF1792 (5); and C-terminal dGT1 (6) were subjected to western blotting analysis with Fap1-peptide specific mAbE42 (top) and anti-DNAK antibody (bottom)as a sample loading control. Complementation of DUF1792 produces an intermediate of Fap1, while the complementation with the C-terminus alone has the same phenotype as dGT1 mutant.(c)The DXE motif and UDP binding sites are highly conserved in bacteria. DUF1792 homologs were identified from a group of streptococci, Lactobacillus lactis and several Gram-negative bacteria. The regions flanking the DGE motif, and UDP binding sites were compared. The invariant amino acid residues highlighted in red and consensus sequence was deduced.

Mentions: To further define the function of DUF1792, we examined the ability of DUF1792 to catalyze the third step of Fap1 glycosylation using a well-established E. coli glycosylation system35. Since we have demonstrated that Gtf1/2 and Gtf3 catalyze the first two steps of Fap1 glycosylation respectively, we co-expressed either DUF1792 or the full-length dGT1 with Gtf1/2, 3 and recombinant Fap1 (rFap1)35 to determine whether dGT1 or DUF1792 further glycosylates the Gtf1/2,3 modified rFap1. Indeed, dGT1 retarded the migration of the Gtf1/2,3 modified rFap1 (Fig. 3a, lane3 versus 2), suggesting additional modification by dGT1. Interestingly, the migration of the modified rFap1 was further retarded when co-expressed with the DUF1792 domain itself (Fig. 3a, lane 4). This is also true for the in vitro glycosyltransferase activity (Fig. 2b). The activity of DUF1792 is consistently higher than that from the full-length dGT1, suggesting the dGT1 C-terminus may have an additional unknown glycosyltransferase activity that coordinates with the function of DUF1792 in vitro. To further determine the relative contribution of DUF1792 and C-terminal dGT1 to Fap1 glycosylation in the native host S. parasanguinis, the dGT1 mutant of S. parasanguinis was complemented by either DUF1792 or C-terminal dGT1, and then examined by Fap1-specific antibody mAbE42. The DUF1792 alone significantly retarded the migration of Fap1 indicative of glycosylation (Fig. 3b, lane 5) in comparison with the dGT1 mutant (Fig. 3b, lane 3) albeit it did not restore the migration as the full-length dGT1 (Fig. 3b, lane 4). By contrast, the C-terminal dGT1 failed to restore the migration, suggesting that the DUF1792 domain is more important than the C-terminal domain in vivo in S. parasanguinis, and that both domains are required for biogenesis of mature Fap1. The detailed function of the C-terminal domain and how it contributes to the Fap1 glycosylation, is under active investigation. DUF1792 is highly conserved in streptococci and several Gram-negative bacteria (Fig. 3c and Supplementary Fig. 2). It is also present in archea (Supplementary Fig. 2). To assess the functional conservation of DUF1792, we selected DUF1792 homologs from other streptococci and a Gram-negative bacterium, Fusobacterium nucleatum to evaluate whether they can further modify the Fap1 glycosylated by Gtf1/2, 3. All DUF1792 homologs (Fig. 3a, lanes 5–9 versus 2) retarded the migration of the Gtf123 modified Fap1, suggesting additional sugar residues were transferred to the Gtf123-modified Fap1.


The highly conserved domain of unknown function 1792 has a distinct glycosyltransferase fold.

Zhang H, Zhu F, Yang T, Ding L, Zhou M, Li J, Haslam SM, Dell A, Erlandsen H, Wu H - Nat Commun (2014)

The glycosyltransferase activity and DXE motif of DUF1792 are conserved(a) DUF1792 is functionally conserved. DUF1792 homologs from a variety of bacterial species were cloned into vector pGEx-6p-1 and co-transformed in E. coli carrying rFap1 and Gtf1/2, 3 to determine the ability of DUF1792 to catalyze the transfer of additional sugar residues to Gtf1/2, 3 modified rFap1. Cell lysates from rFap1 (1), Gtf1/2, 3 modified rFap1 (2) and Gtf1/2, 3 modified rFap1coexpressed with dGT1 of S. parasanguinis (3), or with DUF1792 from S. parasanguinis (4), S. agalactiaeCOH1 (5); S. sanguinis SK36 (6), S. agalactiae 2603 V/R (7), S. pneumoniae TIGR4 (8) and F. nucleatum F0401 (9) were subjected to western blotting analysis with Fap1 specific antibody E42. All DUF1792 homologs retarded the migration of rFap1, suggesting they promoted the transfer of additional sugar residues to Gtf1/2, 3 modified Fap1.(b) Contribution of DUF1792 to glycosylation of Fap1. Wild type dGT1, N-terminal dGT1-DUF1792, C-terminal dGT1 constructs were used to complement the dGT1mutant in S. parasanguinis. Cell lysates from wild type S. parasanguinis (1); Fap1 mutant (2); dGT1 mutant (3); the dGT1 mutant complemented with the dGT1 full-length gene (4), DUF1792 (5); and C-terminal dGT1 (6) were subjected to western blotting analysis with Fap1-peptide specific mAbE42 (top) and anti-DNAK antibody (bottom)as a sample loading control. Complementation of DUF1792 produces an intermediate of Fap1, while the complementation with the C-terminus alone has the same phenotype as dGT1 mutant.(c)The DXE motif and UDP binding sites are highly conserved in bacteria. DUF1792 homologs were identified from a group of streptococci, Lactobacillus lactis and several Gram-negative bacteria. The regions flanking the DGE motif, and UDP binding sites were compared. The invariant amino acid residues highlighted in red and consensus sequence was deduced.
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Figure 3: The glycosyltransferase activity and DXE motif of DUF1792 are conserved(a) DUF1792 is functionally conserved. DUF1792 homologs from a variety of bacterial species were cloned into vector pGEx-6p-1 and co-transformed in E. coli carrying rFap1 and Gtf1/2, 3 to determine the ability of DUF1792 to catalyze the transfer of additional sugar residues to Gtf1/2, 3 modified rFap1. Cell lysates from rFap1 (1), Gtf1/2, 3 modified rFap1 (2) and Gtf1/2, 3 modified rFap1coexpressed with dGT1 of S. parasanguinis (3), or with DUF1792 from S. parasanguinis (4), S. agalactiaeCOH1 (5); S. sanguinis SK36 (6), S. agalactiae 2603 V/R (7), S. pneumoniae TIGR4 (8) and F. nucleatum F0401 (9) were subjected to western blotting analysis with Fap1 specific antibody E42. All DUF1792 homologs retarded the migration of rFap1, suggesting they promoted the transfer of additional sugar residues to Gtf1/2, 3 modified Fap1.(b) Contribution of DUF1792 to glycosylation of Fap1. Wild type dGT1, N-terminal dGT1-DUF1792, C-terminal dGT1 constructs were used to complement the dGT1mutant in S. parasanguinis. Cell lysates from wild type S. parasanguinis (1); Fap1 mutant (2); dGT1 mutant (3); the dGT1 mutant complemented with the dGT1 full-length gene (4), DUF1792 (5); and C-terminal dGT1 (6) were subjected to western blotting analysis with Fap1-peptide specific mAbE42 (top) and anti-DNAK antibody (bottom)as a sample loading control. Complementation of DUF1792 produces an intermediate of Fap1, while the complementation with the C-terminus alone has the same phenotype as dGT1 mutant.(c)The DXE motif and UDP binding sites are highly conserved in bacteria. DUF1792 homologs were identified from a group of streptococci, Lactobacillus lactis and several Gram-negative bacteria. The regions flanking the DGE motif, and UDP binding sites were compared. The invariant amino acid residues highlighted in red and consensus sequence was deduced.
Mentions: To further define the function of DUF1792, we examined the ability of DUF1792 to catalyze the third step of Fap1 glycosylation using a well-established E. coli glycosylation system35. Since we have demonstrated that Gtf1/2 and Gtf3 catalyze the first two steps of Fap1 glycosylation respectively, we co-expressed either DUF1792 or the full-length dGT1 with Gtf1/2, 3 and recombinant Fap1 (rFap1)35 to determine whether dGT1 or DUF1792 further glycosylates the Gtf1/2,3 modified rFap1. Indeed, dGT1 retarded the migration of the Gtf1/2,3 modified rFap1 (Fig. 3a, lane3 versus 2), suggesting additional modification by dGT1. Interestingly, the migration of the modified rFap1 was further retarded when co-expressed with the DUF1792 domain itself (Fig. 3a, lane 4). This is also true for the in vitro glycosyltransferase activity (Fig. 2b). The activity of DUF1792 is consistently higher than that from the full-length dGT1, suggesting the dGT1 C-terminus may have an additional unknown glycosyltransferase activity that coordinates with the function of DUF1792 in vitro. To further determine the relative contribution of DUF1792 and C-terminal dGT1 to Fap1 glycosylation in the native host S. parasanguinis, the dGT1 mutant of S. parasanguinis was complemented by either DUF1792 or C-terminal dGT1, and then examined by Fap1-specific antibody mAbE42. The DUF1792 alone significantly retarded the migration of Fap1 indicative of glycosylation (Fig. 3b, lane 5) in comparison with the dGT1 mutant (Fig. 3b, lane 3) albeit it did not restore the migration as the full-length dGT1 (Fig. 3b, lane 4). By contrast, the C-terminal dGT1 failed to restore the migration, suggesting that the DUF1792 domain is more important than the C-terminal domain in vivo in S. parasanguinis, and that both domains are required for biogenesis of mature Fap1. The detailed function of the C-terminal domain and how it contributes to the Fap1 glycosylation, is under active investigation. DUF1792 is highly conserved in streptococci and several Gram-negative bacteria (Fig. 3c and Supplementary Fig. 2). It is also present in archea (Supplementary Fig. 2). To assess the functional conservation of DUF1792, we selected DUF1792 homologs from other streptococci and a Gram-negative bacterium, Fusobacterium nucleatum to evaluate whether they can further modify the Fap1 glycosylated by Gtf1/2, 3. All DUF1792 homologs (Fig. 3a, lanes 5–9 versus 2) retarded the migration of the Gtf123 modified Fap1, suggesting additional sugar residues were transferred to the Gtf123-modified Fap1.

Bottom Line: Structural studies, however, have only revealed two distinct glycosyltransferase (GT) folds, GT-A and GT-B.Biochemical studies reveal that the domain is a glucosyltransferase, and it catalyses the transfer of glucose to the branch point of the hexasaccharide O-linked to the serine-rich repeat of the bacterial adhesin, Fap1 of Streptococcus parasanguinis.Thus, DUF1792 represents a new family of glycosyltransferases; therefore, we designate it as a GT-D glycosyltransferase fold.

View Article: PubMed Central - PubMed

Affiliation: Departments of Pediatric Dentistry, Microbiology, Schools of Dentistry and Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.

ABSTRACT
More than 33,000 glycosyltransferases have been identified. Structural studies, however, have only revealed two distinct glycosyltransferase (GT) folds, GT-A and GT-B. Here we report a 1.34-Å resolution X-ray crystallographic structure of a previously uncharacterized 'domain of unknown function' 1792 (DUF1792) and show that the domain adopts a new fold and is required for glycosylation of a family of serine-rich repeat streptococcal adhesins. Biochemical studies reveal that the domain is a glucosyltransferase, and it catalyses the transfer of glucose to the branch point of the hexasaccharide O-linked to the serine-rich repeat of the bacterial adhesin, Fap1 of Streptococcus parasanguinis. DUF1792 homologues from both Gram-positive and Gram-negative bacteria also exhibit the activity. Thus, DUF1792 represents a new family of glycosyltransferases; therefore, we designate it as a GT-D glycosyltransferase fold. As the domain is highly conserved in bacteria and not found in eukaryotes, it can be explored as a new antibacterial target.

Show MeSH
Related in: MedlinePlus