Investigation of the acetylation mechanism by GCN5 histone acetyltransferase

The histone acetylation of post-translational modification can be highly dynamic and play a crucial role in regulating cellular proliferation, survival, differentiation and motility. Of the enzymes that mediate post-translation modifications, the GCN5 of the histone acetyltransferase (HAT) proteins family that add acetyl groups to target lysine residues within histones, has been most extensively studied. According to the mechanism studies of GCN5 related proteins, two key processes, deprotonation and acetylation, must be involved. However, as a fundamental issue, the structure of hGCN5/AcCoA/pH3 remains elusive. Although biological experiments have proved that GCN5 mediates the acetylation process through the sequential mechanism pathway, a dynamic view of the catalytic process and the molecular basis for hGCN5/AcCoA/pH3 are still not available and none of theoretical studies has been reported to other related enzymes in HAT family. To explore the molecular basis for the catalytic mechanism, computational approaches including molecular modeling, molecular dynamic (MD) simulation and quantum mechanics/molecular mechanics (QM/MM) simulation were carried out. The initial hGCN5/AcCoA/pH3 complex structure was modeled and a reasonable snapshot was extracted from the trajectory of a 20 ns MD simulation, with considering post-MD analysis and reported experimental results. Those residues playing crucial roles in binding affinity and acetylation reaction were comprehensively investigated. It demonstrated Glu80 acted as the general base for deprotonation of Lys171 from H3. Furthermore, the two-dimensional QM/MM potential energy surface was employed to study the sequential pathway acetylation mechanism. Energy barriers of addition-elimination reaction in acetylation obtained from QM/MM calculation indicated the point of the intermediate ternary complex. Our study may provide insights into the detailed mechanism for acetylation reaction of GCN5, and has important implications for the discovery of regulators against GCN5 enzymes and related HAT family enzymes.     

Catalytic mechanism of a MYST family histone acetyltransferase

Distinct catalytic mechanisms have been proposed for the Gcn5 and MYST histone acetyltransferase (HAT) families. Gcn5-like HATs utilize an ordered sequential mechanism involving direct nucleophilic attack of the N-epsilon-lysine on the enzyme-bound acetyl-CoA. Recently, MYST enzymes were reported to employ a ping-pong route of catalysis via an acetyl-cysteine intermediate. Here, using the prototypical MYST family member Esa1, and its physiological complex (piccolo NuA4), steady-state kinetic analyses revealed a kinetic mechanism that requires the formation of a ternary complex prior to catalysis, where acetyl-CoA binds first and CoA is the last product released. In the absence of histone acceptor, slow rates of enzyme auto-acetylation (7 x 10(-4) s(-1), or approximately 2500-fold slower than histone acetylation; kcat = 1.6 s(-1)) and of CoA formation (0.0021 s(-1)) were inconsistent with a kinetically competent acetyl-enzyme intermediate. Previously, Cys-304 of Esa1 was the proposed nucleophile that forms an acetyl-cysteine intermediate. Here, mutation of this cysteine (C304A) in Esa1 or within the piccolo NuA4 complex yielded an enzyme that was catalytically indistinguishable from the wild type. Similarly, a pH rate (kcat) analysis of the wild type and C304A revealed an ionization (pKa = 7.6-7.8) that must be unprotonated. Mutation of a conserved active-site glutamate (E338Q) reduced kcat approximately 200-fold at pH 7.5; however, at higher pH, E338Q exhibited nearly wild-type activity. These data are consistent with Glu-338 (general base) activating the N-epsilon-lysine by deprotonation. Together, the results suggest that MYST family HATs utilize a direct-attack mechanism within an Esa1 x acetyl-CoA x histone ternary complex.     

CAT in the HAT: catabolic inhibition by the histone acetyltransferase GCN5

The nuclear hormone receptor coactivator PGC-1alpha is a key regulator of gluconeogenic genes during fasting. In this issue of Cell Metabolism, Puigserver and colleagues (Lerin et al., 2006) report that the histone acetyltransferase GCN5 inhibits gluconeogenesis by acetylating and sequestering PGC-1alpha in nuclear foci.     

Fluorescence analysis of a dynamic loop in the PCAF/GCN5 histone acetyltransferase

PCAF and GCN5 are histone acetyltransferase (HAT) paralogs which play roles in the remodeling of chromatin in health and disease. Previously, a conformationally flexible loop in the catalytic domain had been observed in the X-ray structures of GCN5 in different liganded states. Here, the conformation and dynamics of this PCAF/GCN5 alpha5-beta6 loop was investigated in solution using tryptophan fluorescence. A mutant human PCAF HAT domain (PCAF(Wloop)) was created in which the natural tryptophan (Trp-514) remote from the alpha5-beta6 loop was replaced with tyrosine and a glutamate within the loop (Glu-641) was substituted with tryptophan. This PCAF(Wloop) protein exhibited catalytic parameters within 3-fold of those of the wild-type PCAF catalytic domain, suggesting that the loop mutation was not deleterious for HAT activity. While saturating CoASH induced a 30% quenching of Trp fluorescence in PCAF(Wloop), binding of the high-affinity bisubstrate analogue H3-CoA-20 led to a 2-fold fluorescence increase. These different effects correlate with the different alpha5-beta6 loop conformations seen previously in X-ray structures. On the basis of stopped-flow fluorescence studies, binding of H3-CoA-20 to PCAF(Wloop) proceeds via a rapid association step followed by a slower conformational change involving loop movement. Time-resolved fluorescence measurements support a model in which the alpha5-beta6 loop in the H3-CoA-20-PCAF(Wloop) complex exists in a narrower ensemble of conformations compared to free PCAF(Wloop). The relevance of loop dynamics to PCAF/GCN5 catalysis and substrate specificity are discussed.     

Characterization of a phosphatase 2C protein as an interacting partner of the histone acetyltransferase GCN5 in Arabidopsis

Arabidopsis GCN5 is a major histone acetyltransferase. The mutation of the gene induces pleiotropic effects on plant development, and affects the expression of a large number of genes. The mechanism of action of this protein in controlling plant chromatin structure and genome expression is not understood. In this work, we report the identification of a number of potential protein interacting partners of GCN5 in Arabidopsis. In particular, GCN5 was shown to interact specifically with a phosphatase 2C protein (AtPP2C-6-6). GCN5 phosphorylated by activities in cellular extracts could be dephosphorylated by AtPP2C-6-6 in vitro. Analysis of T-DNA insertion mutants revealed a positive role of AtPP2C-6-6 in salt induction of stress-inducible genes, while the gcn5 mutation seemed to have no effect on the induction but showed up-regulation of a subset of the stress-inducible genes under non-induced conditions. In addition, the gcn5 mutation seriously reduced acetylation of histone H3K14 and H3K27, whereas the T-DNA insertions of the AtPP2C6-6 gene enhanced the acetylation of these lysine residues. Taken together, the present data suggest that AtPP2C-6-6 may function as a negative regulator of GCN5 activity in Arabidopsis.     

Kinetic mechanism of the reaction catalyzed by nuclear histone acetyltransferase from calf thymus

The kinetic mechanism for calf thymus histone acetyltransferase A has been determined from the initial velocity studies. The kinetic patterns at low substrate concentrations suggest that the reaction proceeds via two half-reactions as in a ping-pong pathway with the formation of an acetyl-enzyme intermediate. Such acetyl-enzyme has been isolated and found to be chemically competent. In addition, product inhibition patterns by coenzyme A are consistent with a hybrid ping-pong mechanism. These findings indicate that the acetyltransferase A from calf thymus has two separate and independent binding sites, one for each of the two substrates. Consequently, the mechanism constructed for the acetyltransferase A catalyzed reaction may be described as a double-displacement, two-site ping-pong mechanism.