GCN5-related N-acetyltransferase Research Articles

Abstract 18405: Epidrug Treatment Rescues Proliferation and Differentiation in Human Cardiac Mesenchymal Cells of Type 2 Diabetic Patients: A Case of Epimetabolic Memory

Introduction: The origin of metabolic/epigenetic memory remains elusive. It, however, may be at the basis of chronic diseases and functional alteration associated to ageing and cardiovascular disease. HYPOTHESIS: Diabetes is known to alter cellular metabolism causing defects in tissue/organ regeneration. In this study we asked whether human cardiac stromal cells from diabetic and normoglycaemic voluntary donors revealed differences associated to their original metabolism and whether the alterations could be rescued by epigenetically active drugs. METHODS and RESULTS: D-CMSC were characterized by a reduced proliferation rate, diminished phosphorylation at histone H3 serine 10 (H3S10P), decreased differentiation potential, and premature cellular senescence. A global histone code profiling of D-CMSC revealed that acetylation on histone H3 lysine 9 (H3K9Ac) and lysine 14 (H3K14Ac) was decreased, whereas the trimethylation of histone H3 lysine 9 and lysine 27 significantly increased. These observations were paralleled by a downregulation of the GCN5- related N-acetyltransferases (GNAT) p300/CBP-associated factor and its isoform 5-a general control of amino acid synthesis (GCN5a), determining a relative decreasein total HAT activity. DNA CpG island hypermethylation was detected at promoters of genes involved in cell growth control and genomic stability. Remarkably, treatment with the GNAT proactivator SPV106 restored normal levels of H3K9Ac and H3K14Ac, reduced DNA CpG hypermethylation, and recovered D-CMSC proliferation and differentiation. CONCLUSIONS: This study investigated the diabetes-associated alterations present in cardiac mesenchymal cells (CMSC) obtained from normoglycemic (ND-CMSC) and type 2 diabetic patients (D-CMSC), identifying the histone acetylase (HAT) activator pentadecylidenemalonate 1b (SPV106) as a potential pharmacological intervention to restore cellular function. These results suggest that epigenetic interventions may reverse alterations in human CMSC obtained from diabetic patients.

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.

Double trouble-Buffer selection and His-tag presence may be responsible for nonreproducibility of biomedical experiments.

The availability of purified and active protein is the starting point for the majority of in vitro biomedical, biochemical, and drug discovery experiments. The use of polyhistidine affinity tags has resulted in great increases of the efficiency of the protein purification process, but can negatively affect structure and/or activity measurements. Similarly, buffer molecules may perturb the conformational stability of a protein or its activity. During the determination of the structure of a Gcn5-related N-acetyltransferase (GNAT) from Pseudomonas aeruginosa (PA4794), we found that both HEPES and the polyhistidine affinity tag bind (separately) in the substrate-binding site. In the case of HEPES, the molecule induces conformational changes in the active site, but does not significantly affect enzyme activity. In contrast, the uncleaved His-tag does not induce major conformational changes but acts as a weak competitive inhibitor of peptide substrate. In two other GNAT enzymes, we observed that the presence of the His-tag had a strong influence on the activity of these proteins. The influence of protein preparation on functional studies may affect the reproducibility of experiments in other laboratories, even when changes between protocols seem at first glance to be insignificant. Moreover, the results presented here show how critical it is to adjust the experimental conditions for each protein or family of proteins, and investigate the influence of these factors on protein activity and structure, as they may significantly alter the effectiveness of functional characterization and screening methods. Thus, we show that a polyhistidine tag and the buffer molecule HEPES bind in the substrate-binding site and influence the conformation of the active site and the activity of GNAT acetyltransferases. We believe that such discrepancies can influence the reproducibility of some experiments and therefore could have a significant "ripple effect" on subsequent studies.

Structural and Functional Characterization of the α-Tubulin Acetyltransferase MEC-17

Tubulin protomers undergo an extensive array of post-translational modifications to tailor microtubules to specific tasks. One such modification, the acetylation of lysine 40 of α-tubulin, located in the lumen of microtubules, is associated with stable, long-living microtubule structures. MEC-17 was recently identified as the acetyltransferase that mediates this event. We have determined the crystal structure of the catalytic core of human MEC-17 in complex with its cofactor acetyl-CoA at 1.7Å resolution. The structure reveals that the MEC-17 core adopts a canonical Gcn5-related N-acetyltransferase (GNAT) fold that is decorated with extensive surface loops. An enzymatic analysis of 33 MEC-17 surface mutants identifies hot-spot residues for catalysis and substrate recognition. A large, evolutionarily conserved hydrophobic surface patch that is critical for enzymatic activity is identified, suggesting that specificity is achieved by interactions with the α-tubulin substrate that extend outside of the modified surface loop. An analysis of MEC-17 mutants in Caenorhabditis elegans shows that enzymatic activity is dispensable for touch sensitivity.

The Histone Acetylase Activator Pentadecylidenemalonate 1b Rescues Proliferation and Differentiation in the Human Cardiac Mesenchymal Cells of Type 2 Diabetic Patients

This study investigates the diabetes-associated alterations present in cardiac mesenchymal cells (CMSC) obtained from normoglycemic (ND-CMSC) and type 2 diabetic patients (D-CMSC), identifying the histone acetylase (HAT) activator pentadecylidenemalonate 1b (SPV106) as a potential pharmacological intervention to restore cellular function. D-CMSC were characterized by a reduced proliferation rate, diminished phosphorylation at histone H3 serine 10 (H3S10P), decreased differentiation potential, and premature cellular senescence. A global histone code profiling of D-CMSC revealed that acetylation on histone H3 lysine 9 (H3K9Ac) and lysine 14 (H3K14Ac) was decreased, whereas the trimethylation of H3K9Ac and lysine 27 significantly increased. These observations were paralleled by a downregulation of the GCN5-related N-acetyltransferases (GNAT) p300/CBP-associated factor and its isoform 5-α general control of amino acid synthesis (GCN5a), determining a relative decrease in total HAT activity. DNA CpG island hypermethylation was detected at promoters of genes involved in cell growth control and genomic stability. Remarkably, treatment with the GNAT proactivator SPV106 restored normal levels of H3K9Ac and H3K14Ac, reduced DNA CpG hypermethylation, and recovered D-CMSC proliferation and differentiation. These results suggest that epigenetic interventions may reverse alterations in human CMSC obtained from diabetic patients.

Purification, crystallization and preliminary X-ray diffraction analysis of the N-acetyltransferase SAV0826 from Staphylococcus aureus.

Staphylococcus aureus is a prevalent microorganism that is capable of causing a wide range of infections and diseases. Several strains of this bacterial species have developed antibiotic resistance to methicillin and vancomycin, and higher death rates are still being reported each year owing to multidrug-resistant strains. Certain GCN5-related N-acetyltransferases (GNATs) exhibit a broad substrate range, including aminoglycosides, histones, other proteins and serotonin, and have been implicated in antibiotic drug resistance. Here, the expression, purification, crystallization and preliminary X-ray diffraction analysis of a GNAT from S. aureus (SaNAT) are reported. SaNAT was recombinantly expressed and crystallized by the hanging-drop vapour-diffusion method at 296 K, and the crystals diffracted to 1.7 Å resolution on the MX2 beamline at the Australian Synchrotron. The crystals belonged to space group P43212, with unit-cell parameters a = b = 84.86, c = 49.06 Å, α = β = γ = 90°. A single molecule is likely to be present in the asymmetric unit. A full structural and functional analysis is currently being undertaken to provide novel insights into the protein function, which in turn may provide a basis for drug design.

The Roles of Bacterial GCN5-Related N-acetyltransferases

The GCN5-related N-acetyltransferase (GNAT) superfamily of proteins, widespread in eukaryotes and prokaryotes, can utilize acyl coenzyme A (acyl CoA) to acylate respective acceptor substrates and release both CoA and the acylated products. GNATs have been shown to be involved in multiple physiological events, including bacterial drug resistance, regulation of transcription, stress reaction, and metabolic flux, etc. In the last few years, the importance of GNATs has only emerged in eukaryotes, but bacterial GNATs, particularly those of pathogens, have only recently been explored. In this review, we summarize the main members, structures, inhibitors, and activators of proteins in the GNAT family. We focus on the roles of GNATs in bacteria, particularly Mycobacterium tuberculosis GNATs.

Transcriptomic and genomic evidence for Streptococcus agalactiae adaptation to the bovine environment

BackgroundStreptococcus agalactiae is a major cause of bovine mastitis, which is the dominant health disorder affecting milk production within the dairy industry and is responsible for substantial financial losses to the industry worldwide. However, there is considerable evidence for host adaptation (ecotypes) within S. agalactiae, with both bovine and human sourced isolates showing a high degree of distinctiveness, suggesting differing ability to cause mastitis. Here, we (i) generate RNAseq data from three S. agalactiae isolates (two putative bovine adapted and one human) and (ii) compare publicly available whole genome shotgun sequence data from an additional 202 isolates, obtained from six host species, to elucidate possible genetic factors/adaptations likely important for S. agalactiae growth and survival in the bovine mammary gland.ResultsTests for differential expression showed distinct expression profiles for the three isolates when grown in bovine milk. A key finding for the two putatively bovine adapted isolates was the up regulation of a lactose metabolism operon (Lac.2) that was strongly correlated with the bovine environment (all 36 bovine sourced isolates on GenBank possessed the operon, in contrast to only 8/151 human sourced isolates). Multi locus sequence typing of all genome sequences and phylogenetic analysis using conserved operon genes from 44 S. agalactiae isolates and 16 additional Streptococcus species provided strong evidence for acquisition of the operon via multiple lateral gene transfer events, with all Streptococcus species known to be major causes of mastitis, identified as possible donors. Furthermore, lactose fermentation tests were only positive for isolates possessing Lac.2. Combined, these findings suggest that lactose metabolism is likely an important adaptation to the bovine environment. Additional up regulation in the bovine adapted isolates included genes involved in copper homeostasis, metabolism of purine, pyrimidine, glycerol and glucose, and possibly aminoglycoside antibiotic resistance.ConclusionWe detected several genetic factors likely important in S. agalactiae’s adaptation to the bovine environment, in particular lactose metabolism. Of concern is the up regulation of a putative antibiotic resistance gene (GCN5-related N-acetyltransferase) that might reflect an adaptation to the use of aminoglycoside antibiotics within this environment.

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.

Computational Analysis of N-acetyl transferase in Tribolium castaneum

N-acetyl transferase (NAT) is responsible to catalyze the transfer of acetyl groups to arylamines from acetyl-CoA. Aralkylamine Nacetyl transferase (AANAT), which belongs to GCN5-related N-acetyl transferase member, is a globular 23-kDa cytosolic protein that forms a reversible regulatory complex with 14-3-3 proteins, AANAT regulates the daily cycle of melatonin biosynthesis in mammals, making it an attractive target for therapeutic control of abnormal melatonin production in mood and sleep disorders. There is no evidence available regarding α and β subunits, active site and their ASA value in Dopamine N-acetyl transferase. Therefore, we describe the development of Dopamine N-acetyl transferase model in Tribolium castaneum. We further document the predicted active sites in the structural model with solvent exposed ASA residues. During this study, the model was built by CPH program and validated through PROCHECK, Verify 3D, ERRAT and ProSA for reliability. The active sites were predicted in the model with further ASA analysis of active site residues. The discussed information thus provides insight to the predicted active site and ASA values of Dopamine N-acetyl transferase model in Tribolium castaneum.

Structural and functional analysis of the yeast N -acetyltransferase Mpr1 involved in oxidative stress tolerance via proline metabolism

Mpr1 (sigma1278b gene for proline-analog resistance 1), which was originally isolated as N-acetyltransferase detoxifying the proline analog L-azetidine-2-carboxylate, protects yeast cells from various oxidative stresses. Mpr1 mediates the L-proline and L-arginine metabolism by acetylating L-Δ(1)-pyrroline-5-carboxylate, leading to the L-arginine-dependent production of nitric oxide, which confers oxidative stress tolerance. Mpr1 belongs to the Gcn5-related N-acetyltransferase (GNAT) superfamily, but exhibits poor sequence homology with the GNAT enzymes and unique substrate specificity. Here, we present the X-ray crystal structure of Mpr1 and its complex with the substrate cis-4-hydroxy-L-proline at 1.9 and 2.3 Å resolution, respectively. Mpr1 is folded into α/β-structure with eight-stranded mixed β-sheets and six α-helices. The substrate binds to Asn135 and the backbone amide of Asn172 and Leu173, and the predicted acetyl-CoA-binding site is located near the backbone amide of Phe138 and the side chain of Asn178. Alanine substitution of Asn178, which can interact with the sulfur of acetyl-CoA, caused a large reduction in the apparent kcat value. The replacement of Asn135 led to a remarkable increase in the apparent Km value. These results indicate that Asn178 and Asn135 play an important role in catalysis and substrate recognition, respectively. Such a catalytic mechanism has not been reported in the GNAT proteins. Importantly, the amino acid substitutions in these residues increased the L-Δ(1)-pyrroline-5-carboxylate level in yeast cells exposed to heat stress, indicating that these residues are also crucial for its physiological functions. These studies provide some benefits of Mpr1 applications, such as the breeding of industrial yeasts and the development of antifungal drugs.

Biochemical Characterization of Hpa2 and Hpa3, Two Small Closely Related Acetyltransferases from Saccharomyces cerevisiae

Based on their sequences, the Saccharomyces cerevisiae Hpa2 and Hpa3 proteins are annotated as two closely related members of the Gcn5 acetyltransferase family. Here, we describe the biochemical characterization of Hpa2 and Hpa3 as bona fide acetyltransferases with different substrate specificities. Mutational and MALDI-TOF analyses showed that Hpa3 translation initiates primarily from Met-19 rather than the annotated start site, Met-1, with a minor product starting at Met-27. When expressed in Escherichia coli and assayed in vitro, Hpa2 and Hpa3 (from Met-19) acetylated histones and polyamines. Whereas Hpa2 acetylated histones H3 and H4 (at H3 Lys-14, H4 Lys-5, and H4 Lys-12), Hpa3 acetylated only histone H4 (at Lys-8). Additionally, Hpa2, but not Hpa3, acetylated certain small basic proteins. Hpa3, but not Hpa2, has been reported to acetylate D-amino acids, and we present results consistent with that. Overexpression of Hpa2 or Hpa3 is toxic to yeast cells. However, their deletions do not show any standard phenotypic defects. These results suggest that Hpa2 and Hpa3 are similar but distinct acetyltransferases that might have overlapping roles with other known acetyltransferases in vivo in acetylating histones and other small proteins.

Mab_3168c, a Putative Acetyltransferase, Enhances Adherence, Intracellular Survival and Antimicrobial Resistance of Mycobacterium abscessus

Mycobacterium abscessus is a non-tuberculous mycobacterium. It can cause diseases in both immunosuppressed and immunocompetent patients and is highly resistant to multiple antimicrobial agents. M. abscessus displays two different colony morphology types: smooth and rough morphotypes. Cells with a rough morphotype are more virulent. The purpose of this study was to identify genes responsible for M. abscessus morphotype switching. With transposon mutagenesis, a mutant with a Tn5 inserted into the promoter region of the mab_3168c gene was found to switch its colonies from a rough to a smooth morphotype. This mutant had a higher sliding motility but a lower ability to form biofilms, aggregate in culture, and survive inside macrophages. Results of bioinformatic analyses suggest that the putative Mab_3168c protein is a member of the GCN5-related N-acetyltransferase superfamily. This prediction was supported by the demonstration that the mab_3168c gene conferred M. abscessus and M. smegmatis cells resistance to amikacin. The multiple roles of mab_3168c suggest that it could be a potential target for development of therapeutic regimens to treat diseases caused by M. abscessus.

Discovery of a glycerol 3-phosphate phosphatase reveals glycerophospholipid polar head recycling in Mycobacterium tuberculosis

Functional assignment of enzymes encoded by the Mycobacterium tuberculosis genome is largely incomplete despite recent advances in genomics and bioinformatics. Here, we applied an activity-based metabolomic profiling method to assign function to a unique phosphatase, Rv1692. In contrast to its annotation as a nucleotide phosphatase, metabolomic profiling and kinetic characterization indicate that Rv1692 is a D,L-glycerol 3-phosphate phosphatase. Crystal structures of Rv1692 reveal a unique architecture, a fusion of a predicted haloacid dehalogenase fold with a previously unidentified GCN5-related N-acetyltransferase region. Although not directly involved in acetyl transfer, or regulation of enzymatic activity in vitro, this GCN5-related N-acetyltransferase region is critical for the solubility of the phosphatase. Structural and biochemical analysis shows that the active site features are adapted for recognition of small polyol phosphates, and not nucleotide substrates. Functional assignment and metabolomic studies of M. tuberculosis lacking rv1692 demonstrate that Rv1692 is the final enzyme involved in glycerophospholipid recycling/catabolism, a pathway not previously described in M. tuberculosis.

Crystal Structures of Malonyl-Coenzyme A Decarboxylase Provide Insights into Its Catalytic Mechanism and Disease-Causing Mutations

SummaryMalonyl-coenzyme A decarboxylase (MCD) is found from bacteria to humans, has important roles in regulating fatty acid metabolism and food intake, and is an attractive target for drug discovery. We report here four crystal structures of MCD from human, Rhodopseudomonas palustris, Agrobacterium vitis, and Cupriavidus metallidurans at up to 2.3 Å resolution. The MCD monomer contains an N-terminal helical domain involved in oligomerization and a C-terminal catalytic domain. The four structures exhibit substantial differences in the organization of the helical domains and, consequently, the oligomeric states and intersubunit interfaces. Unexpectedly, the MCD catalytic domain is structurally homologous to those of the GCN5-related N-acetyltransferase superfamily, especially the curacin A polyketide synthase catalytic module, with a conserved His-Ser/Thr dyad important for catalysis. Our structures, along with mutagenesis and kinetic studies, provide a molecular basis for understanding pathogenic mutations and catalysis, as well as a template for structure-based drug design.

αTAT1 is the major α-tubulin acetyltransferase in mice

Post-translational modification of tubulin serves as a powerful means for rapidly adjusting the functional diversity of microtubules. Acetylation of the ε-amino group of K40 in α-tubulin is one such modification that is highly conserved in ciliated organisms. Recently, αTAT1, a Gcn5-related N-acetyltransferase, was identified as an α-tubulin acetyltransferase in Tetrahymena and C. elegans. Here we generate mice with a targeted deletion of Atat1 to determine its function in mammals. Remarkably, we observe a loss of detectable K40 α-tubulin acetylation in these mice across multiple tissues and in cellular structures such as cilia and axons where acetylation is normally enriched. Mice are viable and develop normally, however, the absence of Atat1 impacts upon sperm motility and male mouse fertility, and increases microtubule stability. Thus, αTAT1 has a conserved function as the major α-tubulin acetyltransferase in ciliated organisms and has an important role in regulating subcellular specialization of subsets of microtubules.

Broad-substrate screen as a tool to identify substrates for bacterial Gcn5-related N-acetyltransferases with unknown substrate specificity.

Due to a combination of efforts from individual laboratories and structural genomics centers, there has been a surge in the number of members of the Gcn5-related acetyltransferasesuperfamily that have been structurally determined within the past decade. Although the number of three-dimensional structures is increasing steadily, we know little about the individual functions of these enzymes. Part of the difficulty in assigning functions for members of this superfamily is the lack of information regarding how substrates bind to the active site of the protein. The majority of the structures do not show ligand bound in the active site, and since the substrate-binding domain is not strictly conserved, it is difficult to predict the function based on structure alone. Additionally, the enzymes are capable of acetylating a wide variety of metabolites and many may exhibit promiscuity regarding their ability to acetylate multiple classes of substrates, possibly having multiple functions for the same enzyme. Herein, we present an approach to identify potential substrates for previously uncharacterized members of the Gcn5-related acetyltransferase superfamily using a variety of metabolites including polyamines, amino acids, antibiotics, peptides, vitamins, catecholamines, and other metabolites. We have identified potential substrates for eight bacterial enzymes of this superfamily. This information will be used to further structurally and functionally characterize them.

Molecular cloning of rice serotonin N‐acetyltransferase, the penultimate gene in plant melatonin biosynthesis

Because of the absence of an arylalkylamine N-acetyltransferase (AANAT) homolog in the plant genome, the proposal was made that a GCN5-related N-acetyltransferase superfamily gene (GNAT) could be substituted for AANAT. To clone rice serotonin N-acetyltransferase (SNAT), we expressed 31 rice GNAT cDNAs in Escherichia coli and screened SNAT activity by measuring N-acetyltryptamine after application with 1mm tryptamine. GNAT5 was shown to produce high levels of N-acetyltryptamine in E.coli, suggesting a possible rice SNAT. To confirm SNAT activity, the GNAT5 protein was purified through affinity purification from E.coli culture. The purified recombinant GNAT5 showed high SNAT enzyme activity catalyzing serotonin into N-acetylserotonin. The values for Km and Vmax were 385μm and 282 pmol/min/mg protein, respectively. An in vitro enzyme assay of purified SNAT showed N-acetylserotonin formation to be proportional to enzyme concentration and time, with peak activity at pH 8.8. High substrate concentrations above 1mm serotonin inhibited SNAT activity. Finally, the mRNA level of SNAT was higher in shoots than in roots, but it was expressed constitutively, unlike N-acetylserotonin methyltransferase (ASMT), the terminal enzyme in melatonin synthesis. These results suggest that ASMT rather than SNAT is the rate-limiting enzyme of melatonin biosynthesis in plants.

A positive selection approach identifies residues important for folding of Salmonella enterica Pat, an Nε-lysine acetyltransferase that regulates central metabolism enzymes

In Salmonella enterica, the protein acetyltransferase (Pat) enzyme is part of the sirtuin-dependent acylation/deacylation system (SDPADS) that modulates the activity of several proteins via the acylation of lysine residues critical to their activities. Pat is a ∼98 kDa protein with two distinct domains, an N-terminal acyl-CoA synthetase (NDP-forming) domain (∼700 aa) and a C-terminal acetyltransferase domain (∼160 aa), with homology to proteins of the Gcn5-related N-acetyltransferase (GNAT) superfamily. Although the role of the GNAT-like domain is likely responsible for the catalytic activity of Pat, the role of the N-terminal domain remains unclear. Here we report the use of positive selection for identification of residues critical for Pat enzyme activity. This approach revealed seven residues that, when changed, resulted in drastic loss of Pat activity in vitro which caused a discernable loss-of-function phenotype. Five of the seven residues were located in the N-terminal region of Pat and two were located in the GNAT-like domain. Each single-amino-acid variant had a circular dichroism spectrum that differed from that of the wild-type Pat protein, suggesting that loss of enzymatic activity in the mutant proteins was likely due to an inability to acquire its biologically active fold.

Crystal Structure of Elongator Subcomplex Elp4–6

Elongator is a multiprotein complex composed of two subcomplexes, Elp1-3 and Elp4-6. Elongator is highly conserved between yeast and humans and plays an important role in RNA polymerase II-mediated transcriptional elongation and many other processes, including cytoskeleton organization, exocytosis, and tRNA modification. Here, we determined the crystal structure of the Elp4-6 subcomplex of yeast. The overall structure of Elp4-6 revealed that Elp6 acts as a bridge to assemble Elp4 and Elp5. Detailed structural and sequence analyses revealed that each subunit in the Elp4-6 subcomplex forms a RecA-ATPase-like fold, although it lacks the key sequence signature of ATPases. Site-directed mutagenesis and biochemical analyses indicated that the Elp4-6 subcomplex can assemble into a hexameric ring-shaped structure in vitro and in vivo. Furthermore, GST pulldown assays showed that the ring-shaped assembly of the Elp4-6 subcomplex is important for its specific histone H3 binding. Our results may shed light on the substrate recognition and assembly of the holo-Elongator complex.