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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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Glycosyltransferase activity of DUF1792(a) Diagram of dGT1. dGT1 consists of two domains, an N-terminal DUF1792 and a C-terminal GT-A-like domain (WcaA, glycosyltransferase involved in cell wall biosynthesis).(b) In vitro glycosyltransferase activity of DUF1792. Purified dGlT1, and N-terminal DUF1792 fusion proteins catalyzed the transfer of 3H labeled glucose from an activated donor sugar, UDP-3H glucose to Gtf1/2,3-modified Fap1 in in vitro glycosylation reactions, while the C-terminus of dGT1 GST fusion protein and GST failed to transfer. Heated inactivated proteins were used as negative controls to normalize the transfer activity by CPM.(c) Metal ions are required for glycosyltransferase activity of DUF1792. Divalent metal ions, Mn2+, Mg2+ and Ca2+ (10mM) promoted the transfer of 3H labeled glucose from an activated donor sugar, UDP-3Hglucose to Gtf1/2, 3-modified Fap1 in in vitro glycosylation reactions. The value obtained from three different experiments represent means ± standard errors of the means. Significant differences were indicated by asterisks (** P< 0.01, *** P<0.001).
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Figure 2: Glycosyltransferase activity of DUF1792(a) Diagram of dGT1. dGT1 consists of two domains, an N-terminal DUF1792 and a C-terminal GT-A-like domain (WcaA, glycosyltransferase involved in cell wall biosynthesis).(b) In vitro glycosyltransferase activity of DUF1792. Purified dGlT1, and N-terminal DUF1792 fusion proteins catalyzed the transfer of 3H labeled glucose from an activated donor sugar, UDP-3H glucose to Gtf1/2,3-modified Fap1 in in vitro glycosylation reactions, while the C-terminus of dGT1 GST fusion protein and GST failed to transfer. Heated inactivated proteins were used as negative controls to normalize the transfer activity by CPM.(c) Metal ions are required for glycosyltransferase activity of DUF1792. Divalent metal ions, Mn2+, Mg2+ and Ca2+ (10mM) promoted the transfer of 3H labeled glucose from an activated donor sugar, UDP-3Hglucose to Gtf1/2, 3-modified Fap1 in in vitro glycosylation reactions. The value obtained from three different experiments represent means ± standard errors of the means. Significant differences were indicated by asterisks (** P< 0.01, *** P<0.001).

Mentions: While we have determined the first two steps of Fap1 glycosylation34, 36 the remaining glycosylation steps are unknown. In a search for proteins responsible for the subsequent steps of Fap1 glycosylation we identified dGT1 (previously named GalT1 because of its annotated function; we rename it as dGT1 as it has two functional domains). dGT1 is predicted to be a glycosyltransferase since it possesses a putative GT-A type glycosyltransferase domain at the C-terminus. Interestingly, dGT1 also contains a distinct domain of unknown function DUF1792 at the N-terminus (Fig. 2a). In vitro glycosylation assays revealed that full-length dGT1 has a glucosyltransferase activity, transferring glucose residues to Glc-GlcNAc modified Fap1 (Fig. 2b), suggesting dGT1 is involved in the third step of Fap1 glycosylation. To dissect the individual dGT1 domain(s) involved, we expressed both the N-terminal DUF1792 domain (amino acids 1–272) and the C-terminal domain (amino acids 273–582), and determined their activity. Unexpectedly, the N-terminal DUF1792 domain, but not the predicted C-terminal glycosyltransferase GT-A domain is responsible for the in vitro glucosyltransferase activity (Fig. 2b). Moreover, the glucosyltransferase activity of DUF1792 is dependent on the presence of metal ions (Fig. 2c). Mn2+ maximized the activity. However DUF1792 does not have the classic metal binding motif, DXD, found in GT-A family of glycosyltransferases, suggesting that DUF1792 represents a new type of glycosyltransferase.


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)

Glycosyltransferase activity of DUF1792(a) Diagram of dGT1. dGT1 consists of two domains, an N-terminal DUF1792 and a C-terminal GT-A-like domain (WcaA, glycosyltransferase involved in cell wall biosynthesis).(b) In vitro glycosyltransferase activity of DUF1792. Purified dGlT1, and N-terminal DUF1792 fusion proteins catalyzed the transfer of 3H labeled glucose from an activated donor sugar, UDP-3H glucose to Gtf1/2,3-modified Fap1 in in vitro glycosylation reactions, while the C-terminus of dGT1 GST fusion protein and GST failed to transfer. Heated inactivated proteins were used as negative controls to normalize the transfer activity by CPM.(c) Metal ions are required for glycosyltransferase activity of DUF1792. Divalent metal ions, Mn2+, Mg2+ and Ca2+ (10mM) promoted the transfer of 3H labeled glucose from an activated donor sugar, UDP-3Hglucose to Gtf1/2, 3-modified Fap1 in in vitro glycosylation reactions. The value obtained from three different experiments represent means ± standard errors of the means. Significant differences were indicated by asterisks (** P< 0.01, *** P<0.001).
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Figure 2: Glycosyltransferase activity of DUF1792(a) Diagram of dGT1. dGT1 consists of two domains, an N-terminal DUF1792 and a C-terminal GT-A-like domain (WcaA, glycosyltransferase involved in cell wall biosynthesis).(b) In vitro glycosyltransferase activity of DUF1792. Purified dGlT1, and N-terminal DUF1792 fusion proteins catalyzed the transfer of 3H labeled glucose from an activated donor sugar, UDP-3H glucose to Gtf1/2,3-modified Fap1 in in vitro glycosylation reactions, while the C-terminus of dGT1 GST fusion protein and GST failed to transfer. Heated inactivated proteins were used as negative controls to normalize the transfer activity by CPM.(c) Metal ions are required for glycosyltransferase activity of DUF1792. Divalent metal ions, Mn2+, Mg2+ and Ca2+ (10mM) promoted the transfer of 3H labeled glucose from an activated donor sugar, UDP-3Hglucose to Gtf1/2, 3-modified Fap1 in in vitro glycosylation reactions. The value obtained from three different experiments represent means ± standard errors of the means. Significant differences were indicated by asterisks (** P< 0.01, *** P<0.001).
Mentions: While we have determined the first two steps of Fap1 glycosylation34, 36 the remaining glycosylation steps are unknown. In a search for proteins responsible for the subsequent steps of Fap1 glycosylation we identified dGT1 (previously named GalT1 because of its annotated function; we rename it as dGT1 as it has two functional domains). dGT1 is predicted to be a glycosyltransferase since it possesses a putative GT-A type glycosyltransferase domain at the C-terminus. Interestingly, dGT1 also contains a distinct domain of unknown function DUF1792 at the N-terminus (Fig. 2a). In vitro glycosylation assays revealed that full-length dGT1 has a glucosyltransferase activity, transferring glucose residues to Glc-GlcNAc modified Fap1 (Fig. 2b), suggesting dGT1 is involved in the third step of Fap1 glycosylation. To dissect the individual dGT1 domain(s) involved, we expressed both the N-terminal DUF1792 domain (amino acids 1–272) and the C-terminal domain (amino acids 273–582), and determined their activity. Unexpectedly, the N-terminal DUF1792 domain, but not the predicted C-terminal glycosyltransferase GT-A domain is responsible for the in vitro glucosyltransferase activity (Fig. 2b). Moreover, the glucosyltransferase activity of DUF1792 is dependent on the presence of metal ions (Fig. 2c). Mn2+ maximized the activity. However DUF1792 does not have the classic metal binding motif, DXD, found in GT-A family of glycosyltransferases, suggesting that DUF1792 represents a new type of glycosyltransferase.

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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