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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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Related in: MedlinePlus

Sensorgram for binding of AcCoA to wild-type OgpAT. AcCoA was injected in duplicates at 7 concentrations (0.2, 0.7, 2.1, 6.2, 18.5, 55.5 and 166.7 µM). Equilibrium affinity fit is shown in (b) of the figure. RU, relative units.
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RSOB130021F3: Sensorgram for binding of AcCoA to wild-type OgpAT. AcCoA was injected in duplicates at 7 concentrations (0.2, 0.7, 2.1, 6.2, 18.5, 55.5 and 166.7 µM). Equilibrium affinity fit is shown in (b) of the figure. RU, relative units.

Mentions: SPR was used to investigate OgpAT and hOGA-AT interactions with AcCoA. As expected from the crystallographic complex, wild-type OgpAT binds AcCoA (Kd = 8.7 μM, figure 3 and table 2). By contrast, wild-type hOGA-AT did not show any detectable binding of AcCoA. Additional SPR experiments with butyryl-CoA, decanoyl-CoA and CoA again revealed no detectable binding to hOGA-AT (table 2) in agreement with the structural analysis of the hOGA-AT model. Furthermore, mass spectrometric experiments showed that hOGA-AT is not purified as a complex with AcCoA from E. coli, which would have precluded the detection of AcCoA binding by SPR (see electronic supplementary material, figure S2). To investigate whether the unusual hOGA-AT ‘P-loop’ is compatible with AcCoA binding, key P-loop residues in OgpAT (Gly144, Arg145, Gly146) were mutated to the corresponding residues in hOGA-AT (Asp, Pro, Ser) (figures 1b and 2). No AcCoA binding could be detected with this OgpAT mutant (table 2). The inverse experiment of mutating Asp853, Pro854, Ser855 in hOGA-AT to the OgpAT equivalent (Gly, Arg, Gly) resulted in insoluble protein.Table 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)

Sensorgram for binding of AcCoA to wild-type OgpAT. AcCoA was injected in duplicates at 7 concentrations (0.2, 0.7, 2.1, 6.2, 18.5, 55.5 and 166.7 µM). Equilibrium affinity fit is shown in (b) of the figure. RU, relative units.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

RSOB130021F3: Sensorgram for binding of AcCoA to wild-type OgpAT. AcCoA was injected in duplicates at 7 concentrations (0.2, 0.7, 2.1, 6.2, 18.5, 55.5 and 166.7 µM). Equilibrium affinity fit is shown in (b) of the figure. RU, relative units.
Mentions: SPR was used to investigate OgpAT and hOGA-AT interactions with AcCoA. As expected from the crystallographic complex, wild-type OgpAT binds AcCoA (Kd = 8.7 μM, figure 3 and table 2). By contrast, wild-type hOGA-AT did not show any detectable binding of AcCoA. Additional SPR experiments with butyryl-CoA, decanoyl-CoA and CoA again revealed no detectable binding to hOGA-AT (table 2) in agreement with the structural analysis of the hOGA-AT model. Furthermore, mass spectrometric experiments showed that hOGA-AT is not purified as a complex with AcCoA from E. coli, which would have precluded the detection of AcCoA binding by SPR (see electronic supplementary material, figure S2). To investigate whether the unusual hOGA-AT ‘P-loop’ is compatible with AcCoA binding, key P-loop residues in OgpAT (Gly144, Arg145, Gly146) were mutated to the corresponding residues in hOGA-AT (Asp, Pro, Ser) (figures 1b and 2). No AcCoA binding could be detected with this OgpAT mutant (table 2). The inverse experiment of mutating Asp853, Pro854, Ser855 in hOGA-AT to the OgpAT equivalent (Gly, Arg, Gly) resulted in insoluble protein.Table 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