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. 2009 Jul 17;284(29):19412-9.
doi: 10.1074/jbc.M109.014951. Epub 2009 May 27.

Structure and biochemical characterization of protein acetyltransferase from Sulfolobus solfataricus

Michael M Brent  1 , Ayaka Iwata, Juliana Carten, Kehao Zhao, Ronen Marmorstein
Affiliations

Structure and biochemical characterization of protein acetyltransferase from Sulfolobus solfataricus

Michael M Brent et al. J Biol Chem. .

Abstract

The Sulfolobus solfataricus protein acetyltransferase (PAT) acetylates ALBA, an abundant nonspecific DNA-binding protein, on Lys(16) to reduce its DNA affinity, and the Sir2 deacetylase reverses the modification to cause transcriptional repression. This represents a "primitive" model for chromatin regulation analogous to histone modification in eukaryotes. We report the 1.84-A crystal structure of PAT in complex with coenzyme A. The structure reveals homology to both prokaryotic GNAT acetyltransferases and eukaryotic histone acetyltransferases (HATs), with an additional "bent helix" proximal to the substrate binding site that might play an autoregulatory function. Investigation of active site mutants suggests that PAT does not use a single general base or acid residue for substrate deprotonation and product reprotonation, respectively, and that a diffusional step, such as substrate binding, may be rate-limiting. The catalytic efficiency of PAT toward ALBA is low relative to other acetyltransferases, suggesting that there may be better, unidentified substrates for PAT. The structural similarity of PAT to eukaryotic HATs combined with its conserved role in chromatin regulation suggests that PAT is evolutionarily related to the eukaryotic HATs.

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Figures

FIGURE 1.
FIGURE 1.
Structure of the PAT·CoA complex. A, overall structure of the PAT·CoA complex. The conserved acetyl-CoA binding core region is in yellow, less conserved segments are colored in cyan, and CoA is colored by element. B, PAT-CoA interactions. The electron density is from a simulated annealing omit map contoured at 1.0 σ around the CoA. C, representation of the bent helix (α2) in cyan and the interactions that anchor it proximal to the active site of PAT. CoA is colored by element.
FIGURE 2.
FIGURE 2.
Structural comparison of PAT to other acetyltransferases. A, PAT·CoA complex aligned with S. enterica AAC(6′)-Iy (Protein Data Bank code 1S5K). B, PAT·CoA complex aligned with a tetrahymena Gcn5 ternary complex (PDB code 1PUA) illustrating overlap between the Gcn5 substrate peptide (red) and helix α2 in the active site of PAT.
FIGURE 3.
FIGURE 3.
The PAT active site. A, steady-state kinetic analysis of PAT mutants. Assays were done in duplicate with 833 μm [14C]acetyl-coenzyme A and 3 mm ALBA peptide. Bars represent the activity of each mutant measured in counts and plotted as a percent of wild type PAT counts. Error bars represent the range for the two measurements. B, the putative substrate binding face of PAT showing some of the residues mutated. The position of the general bases of yeast Gcn5 (green, Glu173 from PDB code 1YGH) and AANAT (gray, His120 and His122 from PDB code 1CJW) are shown following superposition of the structures with the PAT·CoA complex. C, pH-rate profile for PAT. Rates represent the mean of two measurements. The pH of each assay buffer was measured at 75 °C to account for the shift in pH at high temperature. D, the active site of PAT highlighting hydrophobic residues Leu79, Phe112, and Met121 around the sulfur of CoA.
FIGURE 4.
FIGURE 4.
The putative substrate binding surface of PAT. Access to the active site is shown in a surface representation with the electrostatic surface prepared using CCP4mg. Red represents negative charge and blue represents positive charge at physiological pH.
FIGURE 5.
FIGURE 5.
Comparison of PAT with eukaryotic HATs. The HATs Gcn5, Esa1, p300, and Rtt109 are shown. Their shared CoA binding core is highlighted in yellow with the surrounding structures shown in green.
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