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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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The OgpAT-AcCoA complex is compared with the Naa50p-CoA-peptide complex [43] and a model of hOGA-AT in complex with a superimposed AcCoA, using stick model views of the active site (a) and molecular surfaces (b). CoA/AcCoA are shown as sticks with green carbon atoms. Red spheres represent water molecules. Hydrogen bonds are shown as black dotted lines. The OgpAT surface is coloured by similarity to hOGA-AT (identical residues = dark blue; chemically similar residues = light blue, figure 1b).
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RSOB130021F2: The OgpAT-AcCoA complex is compared with the Naa50p-CoA-peptide complex [43] and a model of hOGA-AT in complex with a superimposed AcCoA, using stick model views of the active site (a) and molecular surfaces (b). CoA/AcCoA are shown as sticks with green carbon atoms. Red spheres represent water molecules. Hydrogen bonds are shown as black dotted lines. The OgpAT surface is coloured by similarity to hOGA-AT (identical residues = dark blue; chemically similar residues = light blue, figure 1b).

Mentions: Inspection of the putative AcCoA-binding site revealed well-defined /Fo/–/Fc/, ϕcalc electron density for the ligand, allowing building and refinement of the complete AcCoA molecule (figures 1c and 2). The interactions between AcCoA and OgpAT are similar to those observed throughout the GNAT superfamily [43]. The adenosine moiety of AcCoA is located on the OgpAT surface and stacks against helix α6, while the ribose and 3′-phosphate project into the solvent (figure 2). The 3′-phosphate forms a hydrogen bond interaction with the side chain of His184 located at the end of helix α6. The pyrophosphate and pantetheine moieties form a series of both direct and water-mediated hydrogen bonds to the protein (figure 2). The most conserved interactions between the protein and AcCoA involve the ‘P-loop’ motif [38], which resides at the beginning of helix α5 (figures 1c and 2). The ‘P-loop’, which is conserved within the GNAT AT ‘motif A’ [10,38], is crucial for the recognition of the AcCoA pyrophosphate group in all ATs and consists of a conserved sequence [Gln/Arg]-x-x-Gly-x-[Gly/Ala]. In the OgpAT-AcCoA complex, the ‘P-loop’ is located at the start of helix α5 (figures 1b,c and 2) and consists of residues 143–148 (sequence Gln-Gly-Arg-Gly-Val-Gly). These residues, together with water molecules, form a network of hydrogen bonds with the pyrophosphate group of AcCoA (figure 2).Figure 2.


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)

The OgpAT-AcCoA complex is compared with the Naa50p-CoA-peptide complex [43] and a model of hOGA-AT in complex with a superimposed AcCoA, using stick model views of the active site (a) and molecular surfaces (b). CoA/AcCoA are shown as sticks with green carbon atoms. Red spheres represent water molecules. Hydrogen bonds are shown as black dotted lines. The OgpAT surface is coloured by similarity to hOGA-AT (identical residues = dark blue; chemically similar residues = light blue, figure 1b).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

RSOB130021F2: The OgpAT-AcCoA complex is compared with the Naa50p-CoA-peptide complex [43] and a model of hOGA-AT in complex with a superimposed AcCoA, using stick model views of the active site (a) and molecular surfaces (b). CoA/AcCoA are shown as sticks with green carbon atoms. Red spheres represent water molecules. Hydrogen bonds are shown as black dotted lines. The OgpAT surface is coloured by similarity to hOGA-AT (identical residues = dark blue; chemically similar residues = light blue, figure 1b).
Mentions: Inspection of the putative AcCoA-binding site revealed well-defined /Fo/–/Fc/, ϕcalc electron density for the ligand, allowing building and refinement of the complete AcCoA molecule (figures 1c and 2). The interactions between AcCoA and OgpAT are similar to those observed throughout the GNAT superfamily [43]. The adenosine moiety of AcCoA is located on the OgpAT surface and stacks against helix α6, while the ribose and 3′-phosphate project into the solvent (figure 2). The 3′-phosphate forms a hydrogen bond interaction with the side chain of His184 located at the end of helix α6. The pyrophosphate and pantetheine moieties form a series of both direct and water-mediated hydrogen bonds to the protein (figure 2). The most conserved interactions between the protein and AcCoA involve the ‘P-loop’ motif [38], which resides at the beginning of helix α5 (figures 1c and 2). The ‘P-loop’, which is conserved within the GNAT AT ‘motif A’ [10,38], is crucial for the recognition of the AcCoA pyrophosphate group in all ATs and consists of a conserved sequence [Gln/Arg]-x-x-Gly-x-[Gly/Ala]. In the OgpAT-AcCoA complex, the ‘P-loop’ is located at the start of helix α5 (figures 1b,c and 2) and consists of residues 143–148 (sequence Gln-Gly-Arg-Gly-Val-Gly). These residues, together with water molecules, form a network of hydrogen bonds with the pyrophosphate group of AcCoA (figure 2).Figure 2.

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