Structural,Functional, and Inhibition Studies of a Gcn5-related N-Acetyltransferase (GNAT) Superfamily Protein PA4794: A NEW C-TERMINAL LYSINE PROTEIN ACETYLTRANSFERASE FROM PSEUDOMONAS AERUGINOSA*

The Gcn5-related N-acetyltransferase (GNAT) superfamily is a large group of evolutionarily related acetyltransferases, with multiple paralogs in organisms from all kingdoms of life. The functionally characterized GNATs have been shown to catalyze the transfer of an acetyl group from acetyl-coenzyme A (Ac-CoA) to the amine of a wide range of substrates, including small molecules and proteins. GNATs are prevalent and implicated in a myriad of aspects of eukaryotic and prokaryotic physiology, but functions of many GNATs remain unknown. In this work, we used a multi-pronged approach of x-ray crystallography and biochemical characterization to elucidate the sequence-structure-function relationship of the GNAT superfamily member PA4794 from Pseudomonas aeruginosa. We determined that PA4794 acetylates the Nϵ amine of a C-terminal lysine residue of a peptide, suggesting it is a protein acetyltransferase specific for a C-terminal lysine of a substrate protein or proteins. Furthermore, we identified a number of molecules, including cephalosporin antibiotics, which are inhibitors of PA4794 and bind in its substrate-binding site. Often, these molecules mimic the conformation of the acetylated peptide product. We have determined structures of PA4794 in the apo-form, in complexes with Ac-CoA, CoA, several antibiotics and other small molecules, and a ternary complex with the products of the reaction: CoA and acetylated peptide. Also, we analyzed PA4794 mutants to identify residues important for substrate binding and catalysis.     

Classification and phylogeny for the annotation of novel eukaryotic GNAT acetyltransferases

The enzymes of the GCN5-related N-acetyltransferase (GNAT) superfamily count more than 870 000 members through all kingdoms of life and share the same structural fold. GNAT enzymes transfer an acyl moiety from acyl coenzyme A to a wide range of substrates including aminoglycosides, serotonin, glucosamine-6-phosphate, protein N-termini and lysine residues of histones and other proteins. The GNAT subtype of protein N-terminal acetyltransferases (NATs) alone targets a majority of all eukaryotic proteins stressing the omnipresence of the GNAT enzymes. Despite the highly conserved GNAT fold, sequence similarity is quite low between members of this superfamily even when substrates are similar. Furthermore, this superfamily is phylogenetically not well characterized. Thus functional annotation based on sequence similarity is unreliable and strongly hampered for thousands of GNAT members that remain biochemically uncharacterized. Here we used sequence similarity networks to map the sequence space and propose a new classification for eukaryotic GNAT acetyltransferases. Using the new classification, we built a phylogenetic tree, representing the entire GNAT acetyltransferase superfamily. Our results show that protein NATs have evolved more than once on the GNAT acetylation scaffold. We use our classification to predict the function of uncharacterized sequences and verify by in vitro protein assays that two fungal genes encode NAT enzymes targeting specific protein N-terminal sequences, showing that even slight changes on the GNAT fold can lead to change in substrate specificity. In addition to providing a new map of the relationship between eukaryotic acetyltransferases the classification proposed constitutes a tool to improve functional annotation of GNAT acetyltransferases.     

Structural Characterization of a Gcn5-Related N-Acetyltransferase from Staphylococcus aureus

The Gcn5-related N-acetyltransferases (GNATs) are ubiquitously expressed in nature and perform a diverse range of cellular functions through the acetylation of small molecules and protein substrates. Using activated acetyl coenzyme A as a common acetyl donor, GNATs catalyse the transfer of an acetyl group to acceptor molecules including aminoglycoside antibiotics, glucosamine-6-phosphate, histones, serotonin and spermidine. There is often only very limited sequence conservation between members of the GNAT superfamily, in part, reflecting their capacity to bind a diverse array of substrates. In contrast, the secondary and tertiary structures are highly conserved, but then at the quaternary level there is further diversity, with GNATs shown to exist in monomeric, dimeric, or tetrameric states. Here we describe the X-ray crystallographic structure of a GNAT enzyme from Staphyloccocus aureus with only low sequence identity to previously solved GNAT proteins. It contains many of the classical GNAT motifs, but lacks other hallmarks of the GNAT fold including the classic β-bulge splayed at the β-sheet interface. The protein is likely to be a dimer in solution based on analysis of the asymmetric unit within the crystal structure, homology with related GNAT family members, and size exclusion chromatography. The study provides the first high resolution structure of this enzyme, providing a strong platform for substrate and cofactor modelling, and structural/functional comparisons within this diverse enzyme superfamily.     

cAMP-regulated Protein Lysine Acetylases in Mycobacteria

Cyclic AMP synthesized by Mycobacterium tuberculosis has been shown to play a role in pathogenesis. However, the high levels of intracellular cAMP found in both pathogenic and non-pathogenic mycobacteria suggest that additional and important biological processes are regulated by cAMP in these organisms. We describe here the biochemical characterization of novel cAMP-binding proteins in M. smegmatis and M. tuberculosis (MSMEG_5458 and Rv0998, respectively) that contain a cyclic nucleotide binding domain fused to a domain that shows similarity to the GNAT family of acetyltransferases. We detect protein lysine acetylation in mycobacteria and identify a universal stress protein (USP) as a substrate of MSMEG_5458. Acetylation of a lysine residue in USP is regulated by cAMP, and using a strain deleted for MSMEG_5458, we show that USP is indeed an in vivo substrate for MSMEG_5458. The Rv0998 protein shows a strict cAMP-dependent acetylation of USP, despite a lower affinity for cAMP than MSMEG_5458. Thus, this report not only represents the first demonstration of protein lysine acetylation in mycobacteria but also describes a unique functional interplay between a cyclic nucleotide binding domain and a protein acetyltransferase.     

Prediction of structure and functional residues for O-GlcNAcase,a divergent homologue of acetyltransferases

N-Acetyl-beta-D-glucosaminidase (O-GlcNAcase) is a key enzyme in the posttranslational modification of intracellular proteins by O-linked N-acetylglucosamine (O-GlcNAc). Here, we show that this protein contains two catalytic domains, one homologous to bacterial hyaluronidases and one belonging to the GCN5-related family of acetyltransferases (GNATs). Using sequence and structural information, we predict that the GNAT homologous region contains the O-GlcNAcase activity. Thus, O-GlcNAcase is the first member of the GNAT family not involved in transfer of acetyl groups, adding a new mode of evolution to this large protein family. Comparison with solved structures of different GNATs led to a reliable structure prediction and mapping of residues involved in binding of the GlcNAc-modified proteins and catalysis.     

Crystal structure of the histone acetyltransferase Hpa2: A tetrameric member of the Gcn5-related N-acetyltransferase superfamily

We report the crystal structure of the yeast protein Hpa2 in complex with acetyl coenzyme A (AcCoA) at 2.4 A resolution and without cofactor at 2.9 A resolution. Hpa2 is a member of the Gcn5-related N-acetyltransferase (GNAT) superfamily, a family of enzymes with diverse substrates including histones, other proteins, arylalkylamines and aminoglycosides. In vitro, Hpa2 is able to acetylate specific lysine residues of histones H3 and H4 with a preference for Lys14 of histone H3. Hpa2 forms a stable dimer in solution and forms a tetramer upon binding AcCoA. The crystal structure reveals that the Hpa2 tetramer is stabilized by base-pair interactions between the adenine moieties of the bound AcCoA molecules. These base-pairs represent a novel method of stabilizing an oligomeric protein structure. Comparison of the structure of Hpa2 with those of other GNAT superfamily members illustrates a remarkably conserved fold of the catalytic domain of the GNAT family even though members of this family share low levels of sequence homology. This comparison has allowed us to better define the borders of the four sequence motifs that characterize the GNAT family, including a motif that is not discernable in histone acetyltransferases by sequence comparison alone. We discuss implications of the Hpa2 structure for the catalytic mechanism of the GNAT enzymes and the opportunity for multiple histone tail modification created by the tetrameric Hpa2 structure.     

Quantitative analysis of CBP- and P300-induced histone acetylations in vivo using native chromatin

In vivo, histone tails are involved in numerous interactions, including those with DNA, adjacent histones, and other, nonhistone proteins. The amino termini are also the substrates for a number of enzymes, including histone acetyltransferases (HATs), histone deacetylases, and histone methyltransferases. Traditional biochemical approaches defining the substrate specificity profiles of HATs have been performed using purified histone tails, recombinant histones, or purified mononucleosomes as substrates. It is clear that the in vivo presentation of the substrate cannot be accurately represented by using these in vitro approaches. Because of the difficulty in translating in vitro results into in vivo situations, we developed a novel single-cell HAT assay that provides quantitative measurements of endogenous HAT activity. The HAT assay is performed under in vivo conditions by using the native chromatin structure as the physiological substrate. The assay combines the spatial resolving power of laser scanning confocal microscopy with simple statistical analyses to characterize CREB binding protein (CBP)- and P300-induced changes in global histone acetylation levels at specific lysine residues. Here we show that CBP and P300 exhibit unique substrate specificity profiles, consistent with the developmental and functional differences between the two HATs.     

Identification of lysine acetyltransferase p300 substrates using 4-pentynoyl-coenzyme A and bioorthogonal proteomics

Proteomic studies have identified a plethora of lysine acetylated proteins in eukaryotes and bacteria. Determining the individual lysine acetyltransferases responsible for each protein acetylation mark is crucial for elucidating the underlying regulatory mechanisms, but has been challenging due to limited biochemical methods. Here, we describe the application of a bioorthogonal chemical proteomics method to profile and identify substrates of individual lysine acetyltransferases. Addition of 4-pentynoyl-coenzyme A, an alkynyl chemical reporter for protein acetylation, to cell extracts, together with purified lysine acetyltransferase p300, enabled the fluorescent profiling and identification of protein substrates via Cu(I)-catalyzed alkyne-azide cycloaddition. We identified several known protein substrates of the acetyltransferase p300 as well as the lysine residues that were modified. Interestingly, several new candidate p300 substrates and their sites of acetylation were also discovered using this approach. Our results demonstrate that bioorthogonal chemical proteomics allows the rapid substrate identification of individual protein acetyltransferases in vitro.     

Glucose-6-phosphate as a probe for the glucosamine-6-phosphate N-acetyltransferase Michaelis complex

Glucosamine-6-phosphate N-acetyltransferase (GNA1) catalyses the N-acetylation of d-glucosamine-6-phosphate (GlcN-6P), using acetyl-CoA as an acetyl donor. The product GlcNAc-6P is an intermediate in the biosynthesis UDP-GlcNAc. GNA1 is part of the GCN5-related acetyl transferase family (GNATs), which employ a wide range of acceptor substrates. GNA1 has been genetically validated as an antifungal drug target. Detailed knowledge of the Michaelis complex and trajectory towards the transition state would facilitate rational design of inhibitors of GNA1 and other GNAT enzymes. Using the pseudo-substrate glucose-6-phosphate (Glc-6P) as a probe with GNA1 crystals, we have trapped the first GNAT (pseudo-)Michaelis complex, providing direct evidence for the nucleophilic attack of the substrate amine, and giving insight into the protonation of the thiolate leaving group.     

Crystal structure of tabtoxin resistance protein complexed with acetyl coenzyme A reveals the mechanism for beta-lactam acetylation

Tabtoxin resistance protein (TTR) is an enzyme that renders tabtoxin-producing pathogens, such as Pseudomonas syringae, tolerant to their own phytotoxins. Here, we report the crystal structure of TTR complexed with its natural cofactor, acetyl coenzyme A (AcCoA), to 1.55A resolution. The binary complex forms a characteristic "V" shape for substrate binding and contains the four motifs conserved in the GCN5-related N-acetyltransferase (GNAT) superfamily, which also includes the histone acetyltransferases (HATs). A single-step mechanism is proposed to explain the function of three conserved residues, Glu92, Asp130 and Tyr141, in catalyzing the acetyl group transfer to its substrate. We also report that TTR possesses HAT activity and suggest an evolutionary relationship between TTR and other GNAT members.     

Subunit Composition and Substrate Specificity of a MOF-containing Histone Acetyltransferase Distinct from the Male-specific Lethal (MSL) Complex

Human MOF (MYST1), a member of the MYST (Moz-Ybf2/Sas3-Sas2-Tip60) family of histone acetyltransferases (HATs), is the human ortholog of the Drosophila males absent on the first (MOF) protein. MOF is the catalytic subunit of the male-specific lethal (MSL) HAT complex, which plays a key role in dosage compensation in the fly and is responsible for a large fraction of histone H4 lysine 16 (H4K16) acetylation in vivo. MOF was recently reported to be a component of a second HAT complex, designated the non-specific lethal (NSL) complex (Mendjan, S., Taipale, M., Kind, J., Holz, H., Gebhardt, P., Schelder, M., Vermeulen, M., Buscaino, A., Duncan, K., Mueller, J., Wilm, M., Stunnenberg, H. G., Saumweber, H., and Akhtar, A. (2006) Mol. Cell 21, 811–823). Here we report an analysis of the subunit composition and substrate specificity of the NSL complex. Proteomic analyses of complexes purified through multiple candidate subunits reveal that NSL is composed of nine subunits. Two of its subunits, WD repeat domain 5 (WDR5) and host cell factor 1 (HCF1), are shared with members of the MLL/SET family of histone H3 lysine 4 (H3K4) methyltransferase complexes, and a third subunit, MCRS1, is shared with the human INO80 chromatin-remodeling complex. In addition, we show that assembly of the MOF HAT into MSL or NSL complexes controls its substrate specificity. Although MSL-associated MOF acetylates nucleosomal histone H4 almost exclusively on lysine 16, NSL-associated MOF exhibits a relaxed specificity and also acetylates nucleosomal histone H4 on lysines 5 and 8.     

Histone acetyltransferases and histone deacetylases of Physarum polycephalum

DEAE-Sepharose chromatography of plasmodial extracts of the myxomycete Physarum polycephalum reveals the presence of multiple histone acetyltransferases and histone deacetylases. Five putative histone acetyltransferases and histone deacetylases. Five putative histone acetyltransferase forms with different substrate specificity can be discriminated: one enzyme which acetylates all core histones and four enzymes with a preference for each of H3, H2A, H2B or H4. Two histone deacetylases, HD1 and HD2, can be discriminated. They differ with respect to substrate specificity and pH-dependence. The substrate specificity of histone deacetylases is determined using HPLC-purified individual core histone species. The order of acetylated substrate preference is H2A>>H3≥H4> H2B for HD1, H3>H2A>H4 for HD2, respectively; HD2 is inactive with H2B as substrate.     

Crystal structure of the novel PaiA N-acetyltransferase from Thermoplasma acidophilum involved in the negative control of sporulation and degradative enzyme production

GCN5-related N-acetyltransferases (GNATs) are the most widely distributed acetyltransferase systems among all three domains of life. GNATs appear to be involved in several key processes, including microbial antibiotic resistance, compacting eukaryotic DNA, controlling gene expression, and protein synthesis. Here, we report the crystal structure of a putative GNAT Ta0374 from Thermoplasma acidophilum, a hyperacidophilic bacterium, that has been determined in an apo-form, in complex with its natural ligand (acetyl coenzyme A), and in complex with a product of reaction (coenzyme A) obtained by cocrystallization with spermidine. Sequence and structural analysis reveals that Ta0374 belongs to a novel protein family, PaiA, involved in the negative control of sporulation and degradative enzyme production. The crystal structure of Ta0374 confirms that it binds acetyl coenzyme A in a way similar to other GNATs and is capable of acetylating spermidine. Based on structural and docking analysis, it is expected that Glu53 and Tyr93 are key residues for recognizing spermidine. Additionally, we find that the purification His-Tag in the apo-form structure of Ta0374 prevents binding of acetyl coenzyme A in the crystal, though not in solution, and affects a chain-flip rotation of "motif A" which is the most conserved sequence among canonical acetyltransferases.     

Structure of an acetyl-CoA binding protein from Staphylococcus aureus representing a novel subfamily of GCN5-related N-acetyltransferase-like proteins

We have determined the solution NMR structure of SACOL2532, a putative GCN5-like N-acetyltransferase (GNAT) from Staphylococcus aureus. SACOL2532 was shown to bind both CoA and acetyl-CoA, and structures with and without bound CoA were determined. Based on analysis of the structure and sequence, a subfamily of small GCN5-related N-acetyltransferase (GNAT)-like proteins can be defined. Proteins from this subfamily, which is largely congruent with COG2388, are characterized by a cysteine residue in the acetyl-CoA binding site near the acetyl group, by their small size in relation to other GNATs, by a lack of obvious substrate binding site, and by a distinct conformation of bound CoA in relation to other GNATs. Subfamily members are found in many bacterial and eukaryotic genomes, and in some archaeal genomes. Whereas other GNATs transfer the acetyl group of acetyl-CoA directly to an aliphatic amine, the presence of the conserved cysteine residue suggests that proteins in the COG2388 GNAT-subfamily transfer an acetyl group from acetyl-CoA to one or more presently unidentified aliphatic amines via an acetyl (cysteine) enzyme intermediate. The apparent absence of a substrate-binding region suggests that the substrate is a macromolecule, such as another protein, or that a second protein subunit providing a substrate-binding region must combine with SACOL2532 to make a fully functional N-acetyl transferase.