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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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UDP and metal binding are required for Fap1 glycosylation in vivoWild type and dGT1 site-directed mutant constructs were used to complement the dGT1mutant in S. parasanguinis. Cell lysates from S. parasanguinis (1); Fap1 mutant (2); dGT1 mutant (3); the dGT1 mutant complemented with the dGT1 full-length gene (4), dGT1 (D31A) (5); dGT1(D31E) (6); and dGT1(H223A) (7) were subjected to western blotting analysis with Fap1-peptide specific mAbE42 (a), mature Fap1-specific mAbF51 (b) to determine Fap1 production, and anti-DNAK antibody (c) as a sample loading control.
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Figure 8: UDP and metal binding are required for Fap1 glycosylation in vivoWild type and dGT1 site-directed mutant constructs were used to complement the dGT1mutant in S. parasanguinis. Cell lysates from S. parasanguinis (1); Fap1 mutant (2); dGT1 mutant (3); the dGT1 mutant complemented with the dGT1 full-length gene (4), dGT1 (D31A) (5); dGT1(D31E) (6); and dGT1(H223A) (7) were subjected to western blotting analysis with Fap1-peptide specific mAbE42 (a), mature Fap1-specific mAbF51 (b) to determine Fap1 production, and anti-DNAK antibody (c) as a sample loading control.

Mentions: To determine the requirement of UDP and metal binding sites in vivo in S. parasanguinis, we selected one key residue Asp31 engaged in the metal binding, and another one His223 involved in the UDP binding, to carry out site-directed mutagenesis and determined the impact of the mutated dGT1 alleles on biogenesis of Fap1 in S. parasanguinis. D31A, D31E and H223A completely inhibited the production of mature Fap1 as determined by mature Fap1 specific mAbF51 antibody (Fig. 8a, lanes 5–7). The Fap1 variants generated by the dGT1 site-directed mutants (Fig. 8b, lanes 5–7) show a similar migration pattern to the Fap1 protein from the dGT1 non mutant (Fig. 8b, lane 3) when probed by Fap1-peptide specific antibody mAbE42, further demonstrating the importance of these two motifs in the glycosylation of Fap1 in vivo.


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)

UDP and metal binding are required for Fap1 glycosylation in vivoWild type and dGT1 site-directed mutant constructs were used to complement the dGT1mutant in S. parasanguinis. Cell lysates from S. parasanguinis (1); Fap1 mutant (2); dGT1 mutant (3); the dGT1 mutant complemented with the dGT1 full-length gene (4), dGT1 (D31A) (5); dGT1(D31E) (6); and dGT1(H223A) (7) were subjected to western blotting analysis with Fap1-peptide specific mAbE42 (a), mature Fap1-specific mAbF51 (b) to determine Fap1 production, and anti-DNAK antibody (c) as a sample loading control.
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Related In: Results  -  Collection

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Figure 8: UDP and metal binding are required for Fap1 glycosylation in vivoWild type and dGT1 site-directed mutant constructs were used to complement the dGT1mutant in S. parasanguinis. Cell lysates from S. parasanguinis (1); Fap1 mutant (2); dGT1 mutant (3); the dGT1 mutant complemented with the dGT1 full-length gene (4), dGT1 (D31A) (5); dGT1(D31E) (6); and dGT1(H223A) (7) were subjected to western blotting analysis with Fap1-peptide specific mAbE42 (a), mature Fap1-specific mAbF51 (b) to determine Fap1 production, and anti-DNAK antibody (c) as a sample loading control.
Mentions: To determine the requirement of UDP and metal binding sites in vivo in S. parasanguinis, we selected one key residue Asp31 engaged in the metal binding, and another one His223 involved in the UDP binding, to carry out site-directed mutagenesis and determined the impact of the mutated dGT1 alleles on biogenesis of Fap1 in S. parasanguinis. D31A, D31E and H223A completely inhibited the production of mature Fap1 as determined by mature Fap1 specific mAbF51 antibody (Fig. 8a, lanes 5–7). The Fap1 variants generated by the dGT1 site-directed mutants (Fig. 8b, lanes 5–7) show a similar migration pattern to the Fap1 protein from the dGT1 non mutant (Fig. 8b, lane 3) when probed by Fap1-peptide specific antibody mAbE42, further demonstrating the importance of these two motifs in the glycosylation of Fap1 in vivo.

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