Limits...
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.

Show MeSH

Related in: MedlinePlus

DUF1792 possesses UDP and Manganese binding sites(a) Cross-eyed stereo view showing the electron density of UDP and the manganese ion at the active site. The map shown is a simulated annealing composite omit electron density map calculated in Phenix and contoured at 2.0σ.(b) The UDP binding site. UDP and the amino acid residues involved in UDP binding are labeled and atomic distances are shown in Ångströms.(c) Manganese binding sites. Manganese and amino acid residues involved in manganese binding are shown and labeled. The atomic distances are shown in Ångströms.(d) Critical residues within UDP and metal binding sites required for glycosyltransferase activity of DUF1792. Site-direct mutagenesis was carried out to mutate critical residues that are involved in binding to UDP and Mn2+, the mutant DUF1792 protein variants were assayed for their in vitro glycosyltransferase activity. The value obtained from three different experiments represent means ± standard errors of the means. Significant differences were indicated by asterisks (*** P<0.001).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4352575&req=5

Figure 7: DUF1792 possesses UDP and Manganese binding sites(a) Cross-eyed stereo view showing the electron density of UDP and the manganese ion at the active site. The map shown is a simulated annealing composite omit electron density map calculated in Phenix and contoured at 2.0σ.(b) The UDP binding site. UDP and the amino acid residues involved in UDP binding are labeled and atomic distances are shown in Ångströms.(c) Manganese binding sites. Manganese and amino acid residues involved in manganese binding are shown and labeled. The atomic distances are shown in Ångströms.(d) Critical residues within UDP and metal binding sites required for glycosyltransferase activity of DUF1792. Site-direct mutagenesis was carried out to mutate critical residues that are involved in binding to UDP and Mn2+, the mutant DUF1792 protein variants were assayed for their in vitro glycosyltransferase activity. The value obtained from three different experiments represent means ± standard errors of the means. Significant differences were indicated by asterisks (*** P<0.001).

Mentions: The hallmark of glycosyltransferases is their ability to bind to nucleotide activated sugars47. Our biochemical assays revealed that UDP-glucose is the activated sugar used by DUF1792. In fact, during the crystallization of DUF1792, we found UDP-glucose is critical for recombinant DUF1792 to grow crystals. Presumably the structure of DUF1792 would require the presence of UDP-glucose. However, only UDP was found in the electron density map from DUF1792 (Fig. 5a, c and Fig. 7a), and there was no density for the glucose moiety from UDP-glucose, indicating that the glucose residue may be turned over by in-crystal catalysis. The same phenomenon has been observed for a number of glycosyltransferases using UDP-glucose36, 48 or other activated sugar donors49, 50.


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)

DUF1792 possesses UDP and Manganese binding sites(a) Cross-eyed stereo view showing the electron density of UDP and the manganese ion at the active site. The map shown is a simulated annealing composite omit electron density map calculated in Phenix and contoured at 2.0σ.(b) The UDP binding site. UDP and the amino acid residues involved in UDP binding are labeled and atomic distances are shown in Ångströms.(c) Manganese binding sites. Manganese and amino acid residues involved in manganese binding are shown and labeled. The atomic distances are shown in Ångströms.(d) Critical residues within UDP and metal binding sites required for glycosyltransferase activity of DUF1792. Site-direct mutagenesis was carried out to mutate critical residues that are involved in binding to UDP and Mn2+, the mutant DUF1792 protein variants were assayed for their in vitro glycosyltransferase activity. The value obtained from three different experiments represent means ± standard errors of the means. Significant differences were indicated by asterisks (*** P<0.001).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4352575&req=5

Figure 7: DUF1792 possesses UDP and Manganese binding sites(a) Cross-eyed stereo view showing the electron density of UDP and the manganese ion at the active site. The map shown is a simulated annealing composite omit electron density map calculated in Phenix and contoured at 2.0σ.(b) The UDP binding site. UDP and the amino acid residues involved in UDP binding are labeled and atomic distances are shown in Ångströms.(c) Manganese binding sites. Manganese and amino acid residues involved in manganese binding are shown and labeled. The atomic distances are shown in Ångströms.(d) Critical residues within UDP and metal binding sites required for glycosyltransferase activity of DUF1792. Site-direct mutagenesis was carried out to mutate critical residues that are involved in binding to UDP and Mn2+, the mutant DUF1792 protein variants were assayed for their in vitro glycosyltransferase activity. The value obtained from three different experiments represent means ± standard errors of the means. Significant differences were indicated by asterisks (*** P<0.001).
Mentions: The hallmark of glycosyltransferases is their ability to bind to nucleotide activated sugars47. Our biochemical assays revealed that UDP-glucose is the activated sugar used by DUF1792. In fact, during the crystallization of DUF1792, we found UDP-glucose is critical for recombinant DUF1792 to grow crystals. Presumably the structure of DUF1792 would require the presence of UDP-glucose. However, only UDP was found in the electron density map from DUF1792 (Fig. 5a, c and Fig. 7a), and there was no density for the glucose moiety from UDP-glucose, indicating that the glucose residue may be turned over by in-crystal catalysis. The same phenomenon has been observed for a number of glycosyltransferases using UDP-glucose36, 48 or other activated sugar donors49, 50.

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