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Structure of a bacterial putative acetyltransferase defines the fold of the human O-GlcNAcase C-terminal domain.

Rao FV, Schüttelkopf AW, Dorfmueller HC, Ferenbach AT, Navratilova I, van Aalten DM - Open Biol (2013)

Bottom Line: However, the molecular basis of AT activity of OGA, which has only been reported in vitro, is not presently understood.The structure of OgpAT in complex with acetyl coenzyme A (AcCoA) reveals that, by homology modelling, hOGA-AT adopts a variant AT fold with a unique loop creating a deep tunnel.The structures, together with mutagenesis and surface plasmon resonance data, reveal that while the bacterial OgpAT binds AcCoA, the hOGA-AT does not, as explained by the lack of key residues normally required to bind AcCoA.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Microbiology, University of Dundee, Dow Street, Dundee DD1 5EH, UK.

ABSTRACT
The dynamic modification of proteins by O-linked N-acetylglucosamine (O-GlcNAc) is an essential posttranslational modification present in higher eukaryotes. Removal of O-GlcNAc is catalysed by O-GlcNAcase, a multi-domain enzyme that has been reported to be bifunctional, possessing both glycoside hydrolase and histone acetyltransferase (AT) activity. Insights into the mechanism, protein substrate recognition and inhibition of the hydrolase domain of human OGA (hOGA) have been obtained via the use of the structures of bacterial homologues. However, the molecular basis of AT activity of OGA, which has only been reported in vitro, is not presently understood. Here, we describe the crystal structure of a putative acetyltransferase (OgpAT) that we identified in the genome of the marine bacterium Oceanicola granulosus, showing homology to the hOGA C-terminal AT domain (hOGA-AT). The structure of OgpAT in complex with acetyl coenzyme A (AcCoA) reveals that, by homology modelling, hOGA-AT adopts a variant AT fold with a unique loop creating a deep tunnel. The structures, together with mutagenesis and surface plasmon resonance data, reveal that while the bacterial OgpAT binds AcCoA, the hOGA-AT does not, as explained by the lack of key residues normally required to bind AcCoA. Thus, the C-terminal domain of hOGA is a catalytically incompetent 'pseudo'-AT.

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(a) Location of OgOGA (blue) and OgpAT (red) in the Oceanicola granulosus genome. (b) Sequence alignment of OgpAT and hOGA-AT. Identical residues are depicted in black. Secondary structure (calculated using DSSP [39]) for OgpAT is shown in blue and red for β-strands and α-helices, respectively. Predicted secondary structure elements (calculated using JPred [40] for hOGA-AT are shown in light blue and pink for β-strands and α-helices, respectively. AcCoA-interacting residues of OgpAT are indicated by green squares (interaction involves side chains) or green triangles (interaction involves backbone only). The two magenta boxes represent the two insertions when compared to sequences from other GNAT members. Numbering of the sequences are in accordance with their UniProt entries. Sequences were aligned with ClustaW [41] and annotated using the program ALINE [42]. (c) Cartoon view (colour based on secondary structure) of OgpAT in complex with AcCoA and Naa50p, an N-terminal AT (PDB code 3TFY [43]) in complex with CoA (green carbon atoms). The Naa50P acceptor peptide is shown with black carbon atoms. The two insertion regions in OgpAT are depicted in magenta. The unbiased /Fo/–/Fc/, ϕcalc electron density map for AcCoA is shown in cyan, contoured at 2.5σ.
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RSOB130021F1: (a) Location of OgOGA (blue) and OgpAT (red) in the Oceanicola granulosus genome. (b) Sequence alignment of OgpAT and hOGA-AT. Identical residues are depicted in black. Secondary structure (calculated using DSSP [39]) for OgpAT is shown in blue and red for β-strands and α-helices, respectively. Predicted secondary structure elements (calculated using JPred [40] for hOGA-AT are shown in light blue and pink for β-strands and α-helices, respectively. AcCoA-interacting residues of OgpAT are indicated by green squares (interaction involves side chains) or green triangles (interaction involves backbone only). The two magenta boxes represent the two insertions when compared to sequences from other GNAT members. Numbering of the sequences are in accordance with their UniProt entries. Sequences were aligned with ClustaW [41] and annotated using the program ALINE [42]. (c) Cartoon view (colour based on secondary structure) of OgpAT in complex with AcCoA and Naa50p, an N-terminal AT (PDB code 3TFY [43]) in complex with CoA (green carbon atoms). The Naa50P acceptor peptide is shown with black carbon atoms. The two insertion regions in OgpAT are depicted in magenta. The unbiased /Fo/–/Fc/, ϕcalc electron density map for AcCoA is shown in cyan, contoured at 2.5σ.

Mentions: Despite the efforts by several research groups, full-length and truncated metazoan OGA has so far resisted protein crystallization. Bacterial homologues have previously been used to gain insights into the structure, mechanism and substrate recognition of the metazoan OGA-GH84 catalytic domain [30,32,34]. Of particular interest is a GH84 from the marine bacterium Oceanicola granulosus, OgOGA, which was recently crystallized [34], as it shows higher sequence identity to hOGA when compared with other bacterial OGA homologues, with sequence conservation extending beyond the catalytic core revealing a conserved peptide-binding groove [34]. Strikingly, close inspection of the OgOGA genomic location reveals an open reading frame coding for a predicted AT [25,38] immediately downstream of the OgOGA gene (figure 1a). Sequence alignment of this predicted AT from O. granulosus (OgpAT) with the hOGA-AT domain (hOGA-AT) shows good similarity (30% sequence identity; figure 1b). Furthermore, secondary structure predictions for hOGA-AT and OgpAT using Jpred [40] support that these two domains are structurally similar (figure 1b). Overall, the genomic organization of OgOGA and OgpAT bears remarkable similarity to the domain arrangement in hOGA. The biological functions of OgOGA and OgpAT in O. granulosus are presently unknown and reversible intracellular O-GlcNAc modification of proteins has not been detected in bacteria.Figure 1.


Structure of a bacterial putative acetyltransferase defines the fold of the human O-GlcNAcase C-terminal domain.

Rao FV, Schüttelkopf AW, Dorfmueller HC, Ferenbach AT, Navratilova I, van Aalten DM - Open Biol (2013)

(a) Location of OgOGA (blue) and OgpAT (red) in the Oceanicola granulosus genome. (b) Sequence alignment of OgpAT and hOGA-AT. Identical residues are depicted in black. Secondary structure (calculated using DSSP [39]) for OgpAT is shown in blue and red for β-strands and α-helices, respectively. Predicted secondary structure elements (calculated using JPred [40] for hOGA-AT are shown in light blue and pink for β-strands and α-helices, respectively. AcCoA-interacting residues of OgpAT are indicated by green squares (interaction involves side chains) or green triangles (interaction involves backbone only). The two magenta boxes represent the two insertions when compared to sequences from other GNAT members. Numbering of the sequences are in accordance with their UniProt entries. Sequences were aligned with ClustaW [41] and annotated using the program ALINE [42]. (c) Cartoon view (colour based on secondary structure) of OgpAT in complex with AcCoA and Naa50p, an N-terminal AT (PDB code 3TFY [43]) in complex with CoA (green carbon atoms). The Naa50P acceptor peptide is shown with black carbon atoms. The two insertion regions in OgpAT are depicted in magenta. The unbiased /Fo/–/Fc/, ϕcalc electron density map for AcCoA is shown in cyan, contoured at 2.5σ.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814719&req=5

RSOB130021F1: (a) Location of OgOGA (blue) and OgpAT (red) in the Oceanicola granulosus genome. (b) Sequence alignment of OgpAT and hOGA-AT. Identical residues are depicted in black. Secondary structure (calculated using DSSP [39]) for OgpAT is shown in blue and red for β-strands and α-helices, respectively. Predicted secondary structure elements (calculated using JPred [40] for hOGA-AT are shown in light blue and pink for β-strands and α-helices, respectively. AcCoA-interacting residues of OgpAT are indicated by green squares (interaction involves side chains) or green triangles (interaction involves backbone only). The two magenta boxes represent the two insertions when compared to sequences from other GNAT members. Numbering of the sequences are in accordance with their UniProt entries. Sequences were aligned with ClustaW [41] and annotated using the program ALINE [42]. (c) Cartoon view (colour based on secondary structure) of OgpAT in complex with AcCoA and Naa50p, an N-terminal AT (PDB code 3TFY [43]) in complex with CoA (green carbon atoms). The Naa50P acceptor peptide is shown with black carbon atoms. The two insertion regions in OgpAT are depicted in magenta. The unbiased /Fo/–/Fc/, ϕcalc electron density map for AcCoA is shown in cyan, contoured at 2.5σ.
Mentions: Despite the efforts by several research groups, full-length and truncated metazoan OGA has so far resisted protein crystallization. Bacterial homologues have previously been used to gain insights into the structure, mechanism and substrate recognition of the metazoan OGA-GH84 catalytic domain [30,32,34]. Of particular interest is a GH84 from the marine bacterium Oceanicola granulosus, OgOGA, which was recently crystallized [34], as it shows higher sequence identity to hOGA when compared with other bacterial OGA homologues, with sequence conservation extending beyond the catalytic core revealing a conserved peptide-binding groove [34]. Strikingly, close inspection of the OgOGA genomic location reveals an open reading frame coding for a predicted AT [25,38] immediately downstream of the OgOGA gene (figure 1a). Sequence alignment of this predicted AT from O. granulosus (OgpAT) with the hOGA-AT domain (hOGA-AT) shows good similarity (30% sequence identity; figure 1b). Furthermore, secondary structure predictions for hOGA-AT and OgpAT using Jpred [40] support that these two domains are structurally similar (figure 1b). Overall, the genomic organization of OgOGA and OgpAT bears remarkable similarity to the domain arrangement in hOGA. The biological functions of OgOGA and OgpAT in O. granulosus are presently unknown and reversible intracellular O-GlcNAc modification of proteins has not been detected in bacteria.Figure 1.

Bottom Line: However, the molecular basis of AT activity of OGA, which has only been reported in vitro, is not presently understood.The structure of OgpAT in complex with acetyl coenzyme A (AcCoA) reveals that, by homology modelling, hOGA-AT adopts a variant AT fold with a unique loop creating a deep tunnel.The structures, together with mutagenesis and surface plasmon resonance data, reveal that while the bacterial OgpAT binds AcCoA, the hOGA-AT does not, as explained by the lack of key residues normally required to bind AcCoA.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Microbiology, University of Dundee, Dow Street, Dundee DD1 5EH, UK.

ABSTRACT
The dynamic modification of proteins by O-linked N-acetylglucosamine (O-GlcNAc) is an essential posttranslational modification present in higher eukaryotes. Removal of O-GlcNAc is catalysed by O-GlcNAcase, a multi-domain enzyme that has been reported to be bifunctional, possessing both glycoside hydrolase and histone acetyltransferase (AT) activity. Insights into the mechanism, protein substrate recognition and inhibition of the hydrolase domain of human OGA (hOGA) have been obtained via the use of the structures of bacterial homologues. However, the molecular basis of AT activity of OGA, which has only been reported in vitro, is not presently understood. Here, we describe the crystal structure of a putative acetyltransferase (OgpAT) that we identified in the genome of the marine bacterium Oceanicola granulosus, showing homology to the hOGA C-terminal AT domain (hOGA-AT). The structure of OgpAT in complex with acetyl coenzyme A (AcCoA) reveals that, by homology modelling, hOGA-AT adopts a variant AT fold with a unique loop creating a deep tunnel. The structures, together with mutagenesis and surface plasmon resonance data, reveal that while the bacterial OgpAT binds AcCoA, the hOGA-AT does not, as explained by the lack of key residues normally required to bind AcCoA. Thus, the C-terminal domain of hOGA is a catalytically incompetent 'pseudo'-AT.

Show MeSH
Related in: MedlinePlus