Structure and Biochemical Characterization of Protein Acetyltransferase from Sulfolobus solfataricus

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-Å 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.Sulfolobus solfataricus, a thermoacidophile, is a member of the archaeal domain of life, and is likely to have diverged from bacteria and eukaryotes early during evolution. Despite its lack of a nucleus or other organelles, archaeal DNA replication and chromatin regulation seem to more closely resemble eukaryotes than bacteria (1, 2). Sulfolobus belongs to the phylum Crenarchaeota, which lacks histones, and instead uses two analogous chromatin proteins: Sul7d and ALBA 3 (acetylation lowers binding affinity). Both proteins have been shown to undergo post-translational modification in Sulfolobus. Sul7d is monomethylated (3) and ALBA is acetylated (4, 5). The acetylation of ALBA by protein acetyltransferase (PAT) on Lys 16 has been shown to reduce DNA-binding affinity, and deacetylation of ALBA by archaeal Sir2 deacetylase has been shown to repress transcription in what appears to be a primitive form of chromatin regulation by reversible post-translational modification (4, 5). PAT is also likely to regulate other proteins in Sulfolobus. Based on its homology to PAT from Salmonella enterica, PAT from Sulfolobus may also play a role in metabolism by regulating the activity of acetyl-coenzyme A synthetase (6).There are at least four families of histone acetyltransferases (HATs) in eukaryotes: the Gcn5/PCAF family that also shows sequence and structural homology to the GNAT (Gcn5-related acetyltransferase) superfamily, which includes many small molecule acetyltransferases such as antibiotic acetyltransferases (aminoglycoside N-acetyltransferases) and serotonin N-acetyltransferase; the MYST family, named from the founding members of MOZ, Ybf2/Sas3, Sas2, and Tip60; the metazoan-specific transcriptional coactivators p300 and CREB-binding protein; and the recently characterized fungal-s...

Acylation of Biomolecules in Prokaryotes: a Widespread Strategy for the Control of Biological Function and Metabolic Stress

“…The reader should be cautioned about the "Pat" abbreviation being used to name acetyltransferases that do not belong to the type I, type II, or type III protein acetyltransferases. As a case in point, the Pat enzyme from Sulfolobus solfataricus (SsPat) (PDB accession number 3F8K) is only 160 residues long (73,74). Such a length is substantially shorter than the typical length of type I Pat enzymes, which are between 850 and 1,100 residues long.…”

“…Whatever the role of the N-terminal domain may be, it is likely to also play a role in sensing acetyl-CoA. This conclusion was drawn on the basis of results from isothermal calorimetry experiments, which showed that SePat binds two molecules of acetyl-CoA: one binds to the N-terminal domain, and the other one binds to the catalytic domain (70,(72)(73)(74)(75)(76)(77).…”

“…A subsequent study showed that acetylation of Lys16 decreased the ability of Alba to bind DNA by ϳ3-fold (59). However, those authors determined that Alba is a relatively poor substrate for SsPat and that other substrates may exist in S. solfataricus (74). Lys16 of Alba was shown to be deacetylated by the sirtuin deacetylase in S. solfataricus (Sir2) (200), which increased the affinity of Alba for DNA (200).…”

“…ADP-forming acyl-CoA synthetases have been characterized in archaea and protists (75)(76)(77). In spite of this homology, no catalytic activity has been attributed to the N-terminal domain of type I or type II GNATs (73,74).…”

cAMP-regulated Protein Lysine Acetylases in Mycobacteria

“…5C). In a recent study on the structure of the protein acetyltransferase from Sulfolobus, it was speculated that the process of inserting the lysine residue in a hydrophobic, substrate-binding pocket seen in protein acetyltransferase could significantly alter the pK a of the lysine residue, thereby allowing acetylation to proceed (41). Perhaps a similar mechanism is seen in MSMEG_5458 whereby cAMP binding could alter the hydrophobicity of the substrate binding site, thereby reducing the pK a of the lysine sufficiently to allow the acetylation reaction to occur, without the requirement of the Glu-234 residue to serve as a base (42).…”