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Serotonin N -acetyltransferase (SNAT) is the key rate-limiting enzyme in melatonin biosynthesis. SNAT mediates dual pathways of melatonin biosynthesis in plants by using serotonin and 5-methoxytryptamine (5-MT) as substrates, and a high reaction pH and temperature are essential to its activity. However, little is known of its underlying mechanisms. Herein, we present a detailed reaction mechanism of a SNAT from Oryza sativa through combined structural and molecular dynamics (MD) analysis. We report for the first time the crystal structures of plant SNAT in the apo and binary/ternary complex forms with acetyl-CoA (AcCoA), serotonin, and 5-MT. These structures reveal that Os SNAT exhibits a unique enzymatically active dimeric fold that is not found in all the known structures of arylalkylamine N-acetyltransferase (AANAT) family. The key residues W188, D189, D226, N220, and Y233 located around the active pocket have important role in catalysis which is subsequently confirmed by site-directed mutagenesis. Combined with MD simulations, we hypothesize a novel plausible catalytic mechanism in which D226 and Y233 function as catalytic base and acid during the acetyl-transfer reaction. This work provides a molecular framework for understanding the catalytic mechanisms of plant SNAT and has implications for future protein engineering and biocatalytic applications.

BCCIP was isolated based on its interactions with tumor suppressors BRCA2 and p21. Knockdown or knockout of BCCIP causes embryonic lethality in mice. BCCIP deficient cells exhibit impaired cell proliferation and chromosome instability. BCCIP also plays a key role in biogenesis of ribosome 60S subunits. BCCIP is conserved from yeast to humans, but it has no discernible sequence similarity to proteins of known structures. Here we report two crystal structures of an N-terminal truncated human BCCIPβ, consisting of residues 61-314. Structurally BCCIP is similar to GCN5-related acetyltransferases (GNATs) but contains different sequence motifs. Moreover, both acetyl-CoA and substrate-binding grooves are altered in BCCIP. A large 19-residue flap over the putative CoA binding site adopts either an open or closed conformation in BCCIP. The substrate binding groove is significantly reduced in size and is positively charged despite the acidic isoelectric point of BCCIP. BCCIP has potential binding sites for partner proteins and may have enzymatic activity.

De novo fatty acid biosynthesis in humans is accomplished by a multidomain protein, the Type I fatty acid synthase (FAS). Although ubiquitously expressed in all tissues, fatty acid synthesis is not essential in normal healthy cells due to sufficient supply with fatty acids by the diet. However, FAS is overexpressed in cancer cells and correlates with tumor malignancy, which makes FAS an attractive selective therapeutic target in tumorigenesis. Herein, we present a crystal structure of the condensing part of murine FAS, highly homologous to human FAS, with octanoyl moieties covalently bound to the transferase (MAT—malonyl‐/acetyltransferase) and the condensation (KS—β‐ketoacyl synthase) domain. The MAT domain binds the octanoyl moiety in a novel (unique) conformation, which reflects the pronounced conformational dynamics of the substrate‐binding site responsible for the MAT substrate promiscuity. In contrast, the KS binding pocket just subtly adapts to the octanoyl moiety upon substrate binding. Besides the rigid domain structure, we found a positive cooperative effect in the substrate binding of the KS domain by a comprehensive enzyme kinetic study. These structural and mechanistic findings contribute significantly to our understanding of the mode of action of FAS and may guide future rational inhibitor designs.

The 3-acetyl-1,2-diacylglycerols (acTAGs) are the molecules that are structurally similar to triacylglycerols (TAGs). They are naturally produced by plants of the family Celastraceae and animals such as Cervus nippon and Eurosta solidaginis. The presence of acetate in the sn-3 position of the glycerol backbone confers advantages to these compounds, for example, lower viscosity and calorific value compared to classical TAGs. In this work, the gene EeDAcT, which encodes diacylglycerol acetyltransferase in a species of bush (Euonymus europaeus), was overexpressed in strains Po1d (capable of accumulating storage lipids) and JMY1877 (incapable of accumulating storage lipids) of Yarrowia lipolytica, to test the activity of the gene EeDAcT and the production of acTAGs in oleaginous and nonoleaginous genetic backgrounds. It was observed that both the strains containing the gene EeDAcT (YL33 and YL35 for Po1d and JMY1877 strains, respectively) produced acTAGs. The strain YL33 accumulated up to 20% intracellular lipids, 20% of which was acTAGs, and 40% was TAGs. On the other hand, the strain YL35, which showed interrupted TAGs accumulation, produced up to 10% acTAGs as the only storage lipid. Unfortunately, the quantity of acTAGs produced in YL35 was insignificant, as the overall lipid accumulated in the strain was not more than 4% of the biomass. The fatty acid profile of acTAGs produced by the YL33 strain was remarkably similar to TAGs, and both of these structures were rich in oleic (45%) and palmitic (25%) acids.

While hundreds of consistently altered metabolic genes had been identified in hepatocellular carcinoma (HCC), the prognostic role of them remains to be further elucidated. Messenger RNA expression profiles and clinicopathological data were downloaded from The Cancer Genome Atlas-Liver Hepatocellular Carcinoma and GSE14520 data set from the Gene Expression Omnibus database. Univariate Cox regression analysis and lasso Cox regression model established a novel four-gene metabolic signature (including acetyl-CoA acetyltransferase 1, glutamic-oxaloacetic transaminase 2, phosphatidylserine synthase 2, and uridine-cytidine kinase 2) for HCC prognosis prediction. Patients in the high-risk group shown significantly poorer survival than patients in the low-risk group. The signature was significantly correlated with other negative prognostic factors such as higher α-fetoprotein. The signature was found to be an independent prognostic factor for HCC survival. Nomogram including the signature shown some clinical net benefit for overall survival prediction. Furthermore, gene set enrichment analyses revealed several significantly enriched pathways, which might help explain the underlying mechanisms. Our study identified a novel robust four-gene metabolic signature for HCC prognosis prediction. The signature might reflect the dysregulated metabolic microenvironment and provided potential biomarkers for metabolic therapy and treatment response prediction in HCC.

Enfumafungin is a glycosylated fernene-type triterpenoid produced by the fungus Hormonema carpetanum. Its potent antifungal activity, mediated by its interaction with β-1,3-glucan synthase and the fungal cell wall, has led to its development into the semi-synthetic clinical candidate, ibrexafungerp (=SCY-078). We report on the preliminary identification of the enfumafungin biosynthetic gene cluster (BGC) based on genome sequencing, phylogenetic reconstruction, gene disruption, and cDNA sequencing studies. Enfumafungin synthase (efuA) consists of a terpene cyclase domain (TC) fused to a glycosyltransferase (GT) domain and thus represents a novel multifunctional enzyme. Moreover, the TC domain bears a phylogenetic relationship to bacterial squalene-hopene cyclases (SHC) and includes a typical DXDD motif within the active centre suggesting that efuA evolved from SHCs. Phylogenetic reconstruction of the GT domain indicated that this portion of the fusion gene originated from fungal sterol GTs. Eleven genes flanking efuA are putatively involved in the biosynthesis, regulation, transport and self-resistance of enfumafungin and include an acetyltransferase, three P450 monooxygenases, a dehydrogenase, a desaturase and a reductase. A hypothetical scheme for enfumafungin assembly is proposed in which the E-ring is oxidatively cleaved to yield the four-ring system of enfumafungin. EfuA represents the first member of a widespread lineage of fungal SHCs.

Background Maternal diabetes related neural tube defects (NTDs) are a result of oxidative stress and apoptosis. However, the molecular mechanism behind the pathogenesis is not fully understood. Here, we report that high glucose exposure-induced epigenetic changes influence histone H4 acetylation and neuroepithelial cell proliferation. We also show that the acetyltransferase inhibitor C646 can prevent high glucose induced changes in histone H4 acetylation and neuroepithelial cell proliferation. Methods By using LC-MS/MS as an unbiased approach, we screened the histone acetylation profile in an E9 neuroepithelial cell line (NE-4C) under high glucose exposure. We further explored the mechanism in cells in vitro and in maternal diabetes-induced mouse embryos in vivo. Results We identified 35 core histone acetylation marks in normal E9 neuroepithelial cells, whereas high glucose exposure resulted in novel acetylation sites on H4K31 and H4K44. Acetylation levels of embryonic development associated H4K5/K8/K12/K16 increased in neuroepithelial cells exposed to high glucose in vitro and in brain tissue from maternal diabetes induced exencephalic embryos in vivo. Further, mRNA level of histone acetyltransferase CBP encoded gene Crebbp was significantly increased both in vitro and in vivo. The addition of C646, a selective inhibitor for CBP/p300, significantly rescued increase of H4K5/K8/K12/K16 acetylation levels and H3S10pi-labeled neuroepithelial cell proliferation induced by high glucose exposure. Conclusion Our data provide complementary insights for potential mechanisms of maternal diabetes induced NTDs.

Plants evolved disease resistance (R) proteins that recognize corresponding pathogen effectors and activate effector-triggered immunity (ETI). However, it is largely unknown why, in some cases, a suppressor of ETI exists in plants. Arabidopsis SOBER1 (Suppressor of AvrBsT-elicited Resistance 1) was identified previously as a suppressor of Xanthomonas acetyltransferase effector AvrBsT-triggered immunity. Nevertheless, the extent to which SOBER1 suppresses ETI is unclear. Here, we identified SOBER1 as a suppressor of Pseudomonas acetyltransferase effector HopZ5-triggered immunity in Arabidopsis using recombinant inbred lines. Further analysis showed that SOBER1 suppresses immunity triggered by multiple bacterial acetyltransferases. Interestingly, SOBER1 interferes with the immunity signalling activated by some but not all tested acetyltransferase effectors, indicating that SOBER1 might target components that are shared between several ETI pathways.

Tat-interactive protein 60 consists of an N-terminal chromo barrel domain (TIP60-CB) and a C-terminal acetyltransferase domain and acetylates histone and nonhistone proteins in diverse cellular processes. While TIP60-CB is thought to recognize histone tails, molecular details of this interaction remain unclear. Here, we attempted a quantitative analysis of the interaction between the human TIP60-CB and histone peptides, but did not observe any detectable binding by either fluorescence polarization or isothermal titration calorimetry assays. We also determined the crystal structure of the TIP60-CB alone. Analysis of the apo-structure reveals a putative peptide-binding site that might be occluded by the basic side chain of a residue in a unique β hairpin between the two N-terminal strands of the β barrel, leading to the inability of TIP60-CB to bind histones.

Ubiquitin E3 ligases mediate ubiquitination and degradation of intracellular proteins. We have shown that a relatively new Skp, Cullin, F-box (SCF) protein E3 ligase, SCF FBXL19, has an anti-inflammatory effect and controls actin cytoskeleton dynamics via targeting cell membrane receptor and small GTPases for their ubiquitination and degradation, but the molecular regulation of its subunit FBXL19 stability remains unclear. Here we show that FBXL19 degradation is controlled by the balance between its ubiquitination and acetylation. FBXL19 is an unstable protein with a half-life of ∼3 h. FBXL19 can be polyubiquitinated, and the proteasome inhibitor MG-132 prolongs FBXL19 half-life, suggesting that FBXL19 degradation is mediated in the ubiquitin-proteasome system. FBXL19 can also be acetylated, and enhancing acetylation of FBXL19 by a deacetylase inhibitor reduces FBXL19 ubiquitination levels. Acetylation-mimic FBXL19 mutant exhibits a longer half-life than wild type. An acetyltransferase CBP catalyzes acetylation of FBXL19. Inhibition or down-regulation of CBP reduces FBXL19 stability, whereas it is increased in CBP-overexpressing cells. Taken together, the data indicate that CBP-mediated acetylation reduces ubiquitination and stabilizes FBXL19. Further, we demonstrate that FBXL19 targets small GTPase Cdc42 for its ubiquitination and degradation, whereas this effect is reversed by inhibition of CBP, suggesting that CBP increases the effect of SCF FBXL19 E3 ligase through acetylation and stabilization of FBXL19. Our study reveals a new molecular model for regulation of SCF E3 ligase function by acetylation and stabilization of its subunit F-box protein.-Wei, J., Dong, S., Yao, K., Martinez, M. F. Y. M., Fleisher, P. R., Zhao, Y., Ma, H., Zhao, J. Histone acetyltransferase CBP promotes function of SCF FBXL19 ubiquitin E3 ligase by acetylation and stabilization of its F-box protein subunit.

A type II toxin-antitoxin (TA) system, in which the toxin contains a Gcn5-related N-acetyltransferase (GNAT) domain, has been characterized recently. GNAT toxin acetylates aminoacyl-tRNA and blocks protein translation. It is abolished by the cognate antitoxin that contains the ribbon-helix-helix (RHH) domain. Here, we present an experimental demonstration of the interaction of the GNAT-RHH complex with TA promoter DNA. First, the GNAT-RHH TA locus kacAT was found in Klebsiella pneumoniae HS11286, a strain resistant to multiple antibiotics. Overexpression of KacT halted cell growth and resulted in persister cell formation. The crystal structure also indicated that KacT is a typical acetyltransferase toxin. Co-expression of KacA neutralized KacT toxicity. Expression of the bicistronic kacAT locus was up-regulated during antibiotic stress. Finally, KacT and KacA formed a heterohexamer that interacted with promoter DNA, resulting in negative autoregulation of kacAT transcription. The N-terminus region of KacA accounted for specific binding to the palindromic sequence on the operator DNA, whereas its C-terminus region was essential for the inactivation of the GNAT toxin. These results provide an important insight into the regulation of the GNAT-RHH family TA system.

Protein acetylation is a rapid mechanism for control of protein function. Acetyl-CoA synthetase (AMP-forming, Acs) is the paradigm for the control of metabolic enzymes by lysine acetylation. In many bacteria, type I or II protein acetyltransferases acetylate Acs, however, in actinomycetes type III protein acetyltransferases control the activity of Acs. We measured changes in the activity of the Streptomyces lividans Acs (SlAcs) enzyme upon acetylation by PatB using in vitro and in vivo analyses. In addition to the acetylation of residue K610, residue S608 within the acetylation motif of SlAcs was also acetylated (PKTRSGK610 ). S608 acetylation rendered SlAcs inactive and non-acetylatable by PatB. It is unclear whether acetylation of S608 is enzymatic, but it was clear that this modification occurred in vivo in Streptomyces. In S. lividans, an NAD+ -dependent sirtuin deacetylase from Streptomyces, SrtA (a homologue of the human SIRT4 protein) was needed to maintain SlAcs function in vivo. We have characterized a sirtuin-dependent reversible lysine acetylation system in Streptomyces lividans that targets and controls the Acs enzyme of this bacterium. These studies raise questions about acetyltransferase specificity, and describe the first Acs enzyme in any organism whose activity is modulated by O-Ser and Nɛ -Lys acetylation.

Motility in sperm is driven by the flagella, the principal component of which is the axoneme. The microtubules which make up the 9 + 2 axoneme are composed of heterodimers of alpha and beta tubulins and undergo several post-translational modifications. We have earlier reported that HDAC6 functions as tubulin deacetylase in sperm and has a role in sperm movement. While exploring the specific tubulin acetyltransferase (TAT) in sperm we observed the presence of Chromodomain Y-Like (CDYL), on the principal piece of rat spermatozoa which compelled us to explore its function in sperm. CDYL was observed to be colocalized with acetylated alpha-tubulin (Ac α Tubulin) in sperm flagella. Sperm axonemal fraction showed the presence of CDYL protein indicating its strong association with flagellar microtubules. Sequence alignment of CDYL chromo domain and Alpha tubulin acetyltransferase (αTAT1) revealed that of the 10 residues of αTAT1 known to be involved in α-tubulin binding, 5 residues were identical and 1 was conserved between the two proteins. Docking of CDYL chromo domain and α-tubulin showed that 6 of the 11 important binding residues of α-tubulin showed an interaction with CDYL chromo domain. The putative CDYL chromodomain –α-tubulin interaction was further confirmed by Microscale Thermophoresis. We further asserted the ability of recombinant CDYL and Sperm CDYL to acetylate soluble tubulin and microtubules in vitro. Acetylation of tubulin was increased over two fold in cells overexpressing CDYL. Thus our studies convincingly demonstrate the ability of CDYL to moonlight as a tubulin acetyltransferase. This article is protected by copyright. All rights reserved.

Umu test is one of the in vitro genotoxicity test that has been used widely. It was developed as a high-throughput test system using the 96-well microplate. We have previously constructed new umu test strains for the evaluation of genotoxicity of procarcinogenic metabolic products formed by cytochrome P450 (CYP) enzymes. In this study, a highly sensitive high-throughput genotoxicity test was developed using four umu test strains (OY1002/1A1, OY1002/1B1, OY1002/1A2, and OY1002/3A4) that express human CYPs and NADPH-P450 reductase. We found that the modified umu-microplate method was more sensitive than the conventional microplate method using strain OY1002/1A2. In addition, the new microplate method was better able to detect genotoxicity than the test tube method when the strain OY1002/1A2 was used and had similar sensitivity for the remaining three strains. When the microplate method was used, OY1002/1A2 showed stronger umuC gene expression in the presence of 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole, 2-amino-3-methylimidazo[4,5-f]quinoline, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, 2-aminofluorene, and 2-aminoanthracene compared to other strains. We also confirmed CYP1A2 expression in OY1002/1A2 in this condition. These results indicate that the microplate version of this test system can detect the genotoxicity of heterocyclic and aromatic amines with high sensitivity and can be used for high-throughput screening of potentially genotoxic compounds. Environ. Mol. Mutagen. 58:209-216, 2017. © 2017 Wiley Periodicals, Inc.

Kinetic, thermodynamic, and structural properties of the aminoglycoside N3-acetyltransferase-VIa (AAC-VIa) are determined. Among the aminoglycoside N3-acetyltransferases, AAC-VIa has one of the most limited substrate profiles. Kinetic studies showed that only five aminoglycosides are substrates for this enzyme with a range of fourfold difference in kcat values. Larger differences in KM (∼40-fold) resulted in ∼30-fold variation in kcat /KM . Binding of aminoglycosides to AAC-VIa was enthalpically favored and entropically disfavored with a net result of favorable Gibbs energy (ΔG 55% sequence similarity to the highly promiscuous aminoglycoside N3-acetyltransferase. Proteins 2017; 85:1258-1265. © 2017 Wiley Periodicals, Inc.

Summary Saccharopolyspora erythraea has three AMP-forming acetyl-CoA synthetases (Acs) encoded by acsA1, acsA2, and acsA3. In this work, we found that nitrogen response regulator GlnR can directly interact with the promoter regions of all three genes and can activate their transcription in response to nitrogen availability. The typical GlnR-binding boxes were identified in the promoter regions. Moreover, the activities of three Acs enzymes were modulated by the reversible lysine acetylation (RLA) with acetyltransferase AcuA and NAD+-dependent deacetylase SrtN. Interestingly, GlnR controlled the RLA by directly activating the expression of acuA and srtN. A glnR-deleted mutant (ΔglnR) caused a growth defect in 10 mM acetate minimal medium, a condition under which RLA function is critical to control Acs activity. Overexpression of acuA reversed the growth defect of ΔglnR mutant. Total activity of Acs in cell-free extracts from ΔglnR strain had a 4-fold increase relative to that of wildtype strain. Western Blotting showed that in vivo acetylation levels of Acs were influenced by nitrogen availability and lack of glnR. These results demonstrated that GlnR regulated acetyl-CoA synthetases at transcriptional and post-translational levels, and mediated the interplay between nitrogen and carbon metabolisms by integrating nitrogen signals to modulate the acetate metabolism.

Naa10 is a Nα-terminal acetyltransferase that, in a complex with its auxiliary subunit Naa15, co-translationally acetylates the α-amino group of newly synthetized proteins as they emerge from the ribosome. Roughly 40-50% of the human proteome is acetylated by Naa10, rendering this an enzyme with one of the most broad substrate ranges known. Recently, we reported an X-linked disorder of infancy, Ogden syndrome, in two families harboring a c.109 T > C (p.Ser37Pro) variant in NAA10. In the present study we performed in-depth characterization of a yeast model of Ogden syndrome. Stress tests and proteomic analyses suggest that the S37P mutation disrupts Naa10 function and reduces cellular fitness during heat shock, possibly due to dysregulation of chaperone expression and accumulation. Microarray and RNA-seq revealed a pseudo-diploid gene expression profile in ΔNaa10 cells, likely responsible for a mating defect. In conclusion, the data presented here further support the disruptive nature of the S37P/Ogden mutation and identify affected cellular processes potentially contributing to the severe phenotype seen in Ogden syndrome. Data are available via GEO under identifier GSE86482 or with ProteomeXchange under identifier PXD004923.

Post-translational modification of histones is a crucial mode of transcriptional regulation in eukaryotes. A well-described acetylation modifier of certain lysine residues is the Spt-Ada-Gcn5 acetyltransferase (SAGA) complex assembled around the histone acetyltransferase Gcn5 in Saccharomyces cerevisiae. We identified and characterized the SAGA complex in the rice pathogen Fusarium fujikuroi, well-known for producing a large variety of secondary metabolites (SMs). By using a co-immunoprecipitation approach, almost all of the S. cerevisiae SAGA complex components have been identified, except for the ubiquitinating DUBm module and the chromodomain containing Chd1. Deletion of GCN5 led to impaired growth, loss of conidiation and alteration of SM biosynthesis. Furthermore, we show that Gcn5 is essential for the acetylation of several histone 3 lysines in F. fujikuroi, that is, H3K4, H3K9, H3K18 and H3K27. A genome-wide microarray analysis revealed differential expression of about 30% of the genome with an enrichment of genes involved in primary and secondary metabolism, transport and histone modification. HPLC-based analysis of known SMs revealed significant alterations in the Δgcn5 mutant. While most SM genes were activated by Gcn5 activity, the biosynthesis of the pigment bikaverin was strongly increased upon GCN5 deletion underlining the diverse roles of the SAGA complex in F. fujikuroi.

Bola-amphiphilic surfactants are molecules with fascinating properties. Their unique configuration consisting of a long hydrophobic spacer connecting two hydrophilic entities renders the molecule more water soluble than the average surfactant, but still allows formation of supramolecular structures. These properties make them extremely suitable for applications in in nanotechnology, electronics, and gene and drug delivery. In general, these compounds are obtained by chemical synthesis. We report here an efficient microbial production process for the fully green synthesis of bolaform surfactants. A sophorolipid-producing Starmerella bombicola yeast strain was disabled in its sophorolipid acetyltransferase and lactone esterase, which should logically result in synthesis of non-acetylated acidic sophorolipids; molecules with the classic amphiphilic structure. Surprisingly, also bolaform glycolipids were obtained, with an additional sophorose linked to the free carboxyl end of the acidic sophorolipids as confirmed by MS and NMR analysis. The obtained titers of 27.7 g/L total product are comparable to wild type values, and the novel molecules account for at least 74% of this. Bola-amphiphile biosynthesis proved to be attributed to the promiscuous activity of both UDP-glucosyltransferases UGTA1 and UGTB1 from the core sophorolipid pathway, displaying activity toward non-acetylated intermediates. The absence of acetyl groups seems to trigger formation of bolaform compounds starting from acidic sophorolipids. Hence, wild type S. bombicola produces these compounds only at marginal amounts in general not reaching detection limits. We created a strain knocked-out in its sophorolipid acetyltransferase and lactone esterase able to produce these novel compounds in economical relevant amounts, opening doors for the application of biological-derived bolaform structures. Biotechnol. Bioeng. 2016;113: 2644-2651. © 2016 Wiley Periodicals, Inc.

Acetylation is a core cellular process involved in maintaining genomic integrity, gene regulation, and metabolism. Histone acetyltransferases (HATs) are an enzyme family that regulates these processes by catalyzing the transfer of an acetyl moiety onto target proteins. Perturbations of cellular acetylation profiles have been associated with a variety of disease states, including cancer. Changes in acetylation profiles can be achieved by mechanisms associated with acetyltransferases, such as gene down-regulation or alterations in the activity of key acetyltransferase enzymes. An important set of tools for quantifying enzyme activity are in vitro histone acetylation assays, using either endogenous or tagged overexpressed proteins. Detailed in this unit is an in vitro acetylation assay used to quantify HAT activity. © 2017 by John Wiley & Sons, Inc.

Protein ubiquitination can be reversed by de-ubiquitinating enzymes (DUBs), which are classified into two main classes, cysteine proteases and metalloproteases. Cysteine proteases include ubiquitin-specific proteases (USPs) and ubiquitin C-terminal hydrolases. USP22 is a USP family member and a component of the mammalian Spt-Ada-Gcn5 acetyltransferase transcriptional coactivating complex. Regulator of calcineurin 1 (RCAN1; also known as DSCR1 or MCIP1) functions as an endogenous inhibitor of calcineurin signaling. In the present study, we have identified a novel interaction between USP22 and RCAN1 (RCAN1-1S) in the mammalian cells. In addition, the overexpression of USP22 caused the increase of RCAN1 protein stability. USP22 antagonized the actions of FBW7, NEDD4-2, and β-TrCP E3 ligase on RCAN1 and promoted RCAN1 de-ubiquitination. Moreover, we found that RCAN1 was bound to USP22 in basal conditions, and interferon-α (IFN-α) treatment caused the dissociation of RCAN1 from USP22, which subsequently triggered RCAN1 ubiquitination and proteasome degradation. Taken together, these results suggest that USP22 positively regulates RCAN1 levels, which would consequently affect diverse RCAN1-linked cellular processes, such as the inflammatory process involving the release of IFN-α.

AMP forming acyl-CoA synthetases are members of the ANL Superfamily, a family of adenylate-forming enzymes that are involved in primary and secondary metabolism. This family contains additionally the NRPS adenylation domains as well as beetle luciferases.[1] These enzymes range in size from 50–70 kD and contain a large N-terminal domain of 400–550 residues and a smaller C-terminal domain that is approximately 120 residues in length. The enzymes catalyze two-step reactions that involve the initial adenylation of a carboxylate substrate to form an acyl adenylate. This initial reaction is followed by a second partial reaction, generally transfer of the acyl group from the adenylate to the pantetheine thiol of either CoA or a holo-acyl or peptidyl carrier protein. In contrast, the luciferase enzymes catalyze an oxidative decarboxylation to produce the photoactive product. The enzymes of this family exhibit several interesting features of their structural and biochemical mechanisms. First, the enzymes undergo a process known as domain alternation whereby the smaller C-terminal domain uses a 140° rotation to adopt two distinct catalytic conformations to carryout the two partial reactions. With this rotation, a catalytic lysine that is part of a conserved region that has been designated the A10 motif is transferred into the active site to assist the formation of the adenylate intermediate. Numerous biochemical and structural studies have confirmed this domain alternation for acyl-CoA synthetases[2, 3], NRPS adenylation domains[4, 5], and beetle luciferase enzymes.[6, 7] A second interesting feature of the acyl-CoA synthetases concerns their regulation by post-translational acetylation of the A10 lysine within some family members. The features that dictate recognition of acyl-CoA synthetases by the protein acetyl transferase (PAT) enzymes are unknown and have been the target of structural and biochemical investigation. Acetyl-CoA synthetase from Salmonella enterica, for example, is regulated by acetylation of the catalytic lysine 609.[8] The crystal structure of the native and acetylated enzymes[9] demonstrate no large structural changes that result from the acetylation and the inhibition of the acetylated enzyme is thought to result specifically from the unavailability of the side chain amine of the lysine residue. Recent work[10, 11] has shown that the C-terminal domain of acetyl-CoA synthetase can dictate the PAT interaction. Specifically, Streptomyces lividans contains two closely related enzymes that differ in acetylation susceptibility. The S. lividans acetyl-CoA synthetase (SlAcs) is acetylated inefficiently by the SlPatA, while an acetoacetyl-CoA synthetase (SlAacS) is readily acetylated. Similarly, the SlPatA enzyme is able to recognize and acetylate the Acs enzyme from Salmonella enterica (SeAcs).[10] Studies with chimeric enzymes between SlAcs and SeAcs narrowed down the difference in recognition by the SlPatA acetyltransferase to a stretch of 77 residues from the C-terminal domain. Because of conservation of residues between the two proteins, as few as 41 amino acid residues were responsible for the difference in PatA recognition. The C-terminal domain of members of this adenylate-forming superfamily starts with a two stranded antiparallel β-sheet that has been referred to as the A8 motif, one of 10 conserved sequence regions in this enzyme family.[1] This A8 loop is followed by a small domain composed of a central three stranded β-sheet that is surrounded by several α-helices. Interestingly, many short chain acyl-CoA synthetase enzymes are larger than other superfamily members because of a C-terminal extension. Following a common α-helix at the C-terminus that is shared by all family members, the structures of yeast[12] and bacterial[9] Acs contain an additional helix (Figure 1A). Inspection of sequence databases show that this extension is common to enzyme family members that produce CoA thioesters with short chain acids such as acetate, propionate, and acetoacetate. Unfortunately, the C-terminal extension is not fully resolved in any of the structures published to date. Figure 1 Structure of the SlAacS enzyme To continue our investigation of the structural aspects of domain alternation of the ANL family of enzymes, we have determined the structure of the acetoacetyl-CoA synthetase from S. lividans (SlAacS). The structure in the adenylate-forming conformation and the loop joining the two C-terminal helices is visible. We present here this structure along with an analysis of the C-terminal domain and the regions that have been identified from chimeric studies as potentially playing a role in recognition by the protein acetyl transferase.

Dissolution-dynamic nuclear polarization (dissolution-DNP) for magnetic resonance (MR) spectroscopic imaging has recently emerged as a novel technique for noninvasive studies of the metabolic fate of biomolecules in vivo. Since acetate is the most abundant extra- and intracellular short-chain fatty acid, we focused on [1-(13) C]acetate as a promising candidate for a chemical probe to study the myocardial metabolism of a beating heart. The dissolution-DNP procedure of Na[1-(13) C]acetate for in vivo cardiac applications with a 3 T MR scanner was optimized in pigs during bolus injection of doses of up to 3 mmol. The Na[1-(13) C]acetate formulation was characterized by a liquid-state polarization of 14.2% and a T1Eff in vivo of 17.6 ± 1.7 s. In vivo Na[1-(13) C]acetate kinetics displayed a bimodal shape: [1-(13) C]acetyl carnitine (AcC) was detected in a slice covering the cardiac volume, and the signal of (13) C-acetate and (13) C-AcC was modeled using the total area under the curve (AUC) for kinetic analysis. A good correlation was found between the ratio AUC(AcC)/AUC(acetate) and the apparent kinetic constant of metabolic conversion, from [1-(13) C]acetate to [1-(13) C]AcC (kAcC ), divided by the AcC longitudinal relaxation rate (r1 ). Our study proved the feasibility and the limitations of administration of large doses of hyperpolarized [1-(13) C]acetate to study the myocardial conversion of [1-(13) C]acetate in [1-(13) C]acetyl-carnitine generated by acetyltransferase in healthy pigs.

Fibrosis, the deadly pathological manifestation of an abnormal tissue remodeling in any organ due to excessive collagen deposition, is associated with a wide variety of organ failure-related human diseases. Chronic stress or repeated injury in a particular organ induces abnormal molecular signals that lead to super-activation of matrix protein producing fibroblasts, excessive matrix proteins accumulation, loss of physiological tissue architecture or elasticity, and ultimately leading to organ failure. There is no effective therapy for fibrosis. Factor acetyltransferase p300 (FATp300), a major epigenetic regulator that acetylates specific lysines in histones and transcription factors, is essential for elevated collagen synthesis and the levels of FATp300 are significantly elevated in different fibrotic tissues. Pharmacological inhibition of FAT activity of p300 is associated with decreased collagen synthesis by fibroblasts in tissues and amelioration of organ fibrosis. Therefore, FAT-free p300 is superior for physiological tissue repair and must be exploited as a viable therapeutic target against multi-organ fibrosis.

A recently discovered cause of tuberculosis resistance to a drug of last resort, the aminoglycoside kanamycin, results from modification of this drug by the enhanced intracellular survival (Eis) protein. Eis is a structurally and functionally unique acetyltransferase with an unusual capability of acetylating aminoglycosides at multiple positions. The extent of this regioversatility and its defining protein features are unclear. Herein, we determined the positions and order of acetylation of five aminoglycosides by NMR spectroscopy. This analysis revealed unprecedented acetylation of the 3''-amine of kanamycin, amikacin, and tobramycin, and the γ-amine of the 4-amino-2-hydroxybutyryl group of amikacin. A crystal structure of Eis in complex with coenzyme A and tobramycin revealed how tobramycin can be accommodated in the Eis active site in two binding modes, consistent with its diacetylation. These studies, describing chemical and structural details of acetylation, will guide future efforts towards designing aminoglycosides and Eis inhibitors to overcome resistance in tuberculosis.

Lysine acetylation has emerged as a major post-translational modification involved in diverse cellular functions. Using a combination of immunoisolation and liquid chromatography coupled to accurate mass spectrometry, we determined the first acetylome of the human malaria parasite Plasmodium falciparum during its active proliferation in erythrocytes with 421 acetylation sites identified in 230 proteins. Lysine-acetylated proteins are distributed in the nucleus, cytoplasm, mitochondrion and apicoplast. Whereas occurrence of lysine acetylation in a similarly wide range of cellular functions suggests conservation of lysine acetylation through evolution, the Plasmodium acetylome also revealed significant divergence from those of other eukaryotes and even the closely related parasite Toxoplasma. This divergence is reflected in the acetylation of a large number of Plasmodium-specific proteins and different acetylation sites in evolutionarily conserved acetylated proteins. A prominent example is the abundant acetylation of proteins in the glycolysis pathway but relatively deficient acetylation of enzymes in the citrate cycle. Using specific transgenic lines and inhibitors, we determined that the acetyltransferase PfMYST and lysine deacetylases play important roles in regulating the dynamics of cytoplasmic protein acetylation. The Plasmodium acetylome provides an exciting start point for further exploration of functions of acetylation in the biology of malaria parasites.

Aminoglycoside-modifying enzymes (AGMEs) are expressed in many pathogenic bacteria and cause resistance to aminoglycoside (AG) antibiotics. Remarkably, the substrate promiscuity of AGMEs is quite variable. The molecular basis for such ligand promiscuity is largely unknown as there is not an obvious link between amino acid sequence or structure and the antibiotic profiles of AGMEs. To address this issue, this article presents the first kinetic and thermodynamic characterization of one of the least promiscuous AGMEs, the AG N3 acetyltransferase-IIa (AAC-IIa) and its comparison to two highly promiscuous AGMEs, the AG N3-acetyltransferase-IIIb (AAC-IIIb) and the AG phosphotransferase(3′)-IIIa (APH). Despite having similar antibiotic selectivities, AAC-IIIb and APH catalyze different reactions and share no homology to one another. AAC-IIa and AAC-IIIb catalyze the same reaction and are very similar in both amino acid sequence and structure. However, they demonstrate strong differences in their substrate profiles and kinetic and thermodynamic properties. AAC-IIa and APH are also polar opposites in terms of ligand promiscuity but share no sequence or apparent structural homology. However, they both are highly dynamic and may even contain disordered segments and both adopt well-defined conformations when AGs are bound. Contrary to this AAC-IIIb maintains a well-defined structure even in apo form. Data presented herein suggest that the antibiotic promiscuity of AGMEs may be determined neither by the flexibility of the protein nor the size of the active site cavity alone but strongly modulated or controlled by the effects of the cosubstrate on the dynamic and thermodynamic properties of the enzyme.

The original bacterial two-hybrid system is widely used but does not permit the study of interactions regulated by PTMs. Here, we have built a conditional two-hybrid (C2H) system, in which bait and prey proteins can be co-expressed in the presence of a modifying enzyme such as a methyltransferase, acetyltransferase, or kinase. Any increase or decrease in interaction due to the modification of the proteins can be measured by an increased or decreased level of reporter gene expression. The C2H system is comprised of eight new vectors based on the Novagen Duet co-expression plasmids. These vectors include two multiple cloning sites per vector as well as a hexahistidine tag or S-tag to aid in purification, if desired. We demonstrate the use of the C2H system to study the dimerization of the yeast protein Npl3, which is increased when methylated by the methyltransferase Hmt1.

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.

Regulation of secondary metabolite (SM) gene clusters in Aspergillus nidulans has been shown to occur through cluster-specific transcription factors or through global regulators of chromatin structure such as histone methyltransferases, histone deacetylases, or the putative methyltransferase LaeA. A multicopy suppressor screen for genes capable of returning SM production to the SM deficient ΔlaeA mutant resulted in identification of the essential histone acetyltransferase EsaA, able to complement an esa1 deletion in Saccharomyces cereviseae. Here we report that EsaA plays a novel role in SM cluster activation through histone 4 lysine 12 (H4K12) acetylation in four examined SM gene clusters (sterigmatocystin, penicillin, terrequinone and orsellinic acid), in contrast to no increase in H4K12 acetylation of the housekeeping tubA promoter. This augmented SM cluster acetylation requires LaeA for full effect and correlates with both increased transcript levels and metabolite production relative to wild type. H4K12 levels may thus represent a unique indicator of relative production potential, notably of SMs.

In bacteria, β-alanine is formed via the action of l-aspartate α-decarboxylase (PanD) which is one of the small class of pyruvoyl-dependent enzymes. The pyruvoyl cofactor in these enzymes is formed via the intramolecular rearrangement of a serine residue in the peptide backbone leading to chain cleavage and formation of the covalently-bound cofactor from the serine residue. This reaction was previously thought to be uncatalysed. Here we show that in Escherichia coli, PanD is activated by the putative acetyltransferase YhhK, subsequently termed PanZ. Activation of PanD both in vivo and in vitro is PanZ-dependent. PanZ binds to PanD, and we demonstrate that a PanZ(N45A) site-directed mutant is unable to enhance cleavage of the proenzyme PanD despite retaining affinity for PanD. This suggests that the putative acetyltransferases domain of PanZ may be responsible for activation to enhance the processing of PanD. Although panD is conserved among most bacteria, the panZ gene is conserved only in E. coli-related enterobacterial species including Shigella, Salmonella, Klebsiella and Yersinia. These bacteria are found predominantly in the gut flora where pantothenate is abundant and regulation of PanD by PanZ allows these organisms to closely regulate production of β-alanine and hence pantothenate in response to metabolic demand.

Coenzyme A (CoA) is an essential cofactor for all forms of life. The biochemistry underpinning the assembly of CoA in Escherichia coli and other enterobacteria is well understood, except for the events leading to maturation of the L-aspartate-α-decarboxylase (PanD) enzyme that converts pantothenate to β-alanine. PanD is synthesized as pro-PanD, which undergoes an auto-proteolytic cleavage at residue Ser25 to yield the catalytic pyruvoyl moiety of the enzyme. Since 1990, it has been known that E. coli yhhK strains are pantothenate auxotrophs, but the role of YhhK in pantothenate biosynthesis remained an enigma. Here we show that Salmonella enterica yhhK strains are also pantothenate auxotrophs. In vivo and in vitro evidence shows that YhhK interacts directly with PanD, and that such interactions accelerate pro-PanD maturation. We also show that S. enterica yhhK strains accumulate pro-PanD, and that not all pro-PanD proteins require YhhK for maturation. For example, the Corynebacterium glutamicum panD(+) gene corrected the pantothenate auxotrophy of a S. enterica yhhK strain, supporting in vitro evidence obtained by others that some pro-PanD proteins autocleave at faster rates. We propose the name PanM for YhhK to reflect its role as a trigger of pro-PanD maturation by stabilizing pro-PanD in an autocleavage-prone conformation.

Platelet-activating factor (PAF) is a signaling phospholipid with a significant physiological role in multicellular and unicellular organisms, including fermentative organisms such as yeast. Zymomonas mobilis is an ethanologenic α-proteobacterium currently studied for bioethanol production. In order to examine the presence of PAF and/or PAF inhibitors in Z. mobilis, a new one-step high performance liquid chromatography (HPLC) separation procedure of total lipids was performed, using a C8 reversed-phase semi-preparative column. According to this method and to bioassays based on washed rabbit platelet aggregation, two lipid molecules with PAF-like activity and same retention times as those of standard PAF were detected; electron-spray ionization MS and MS/MS analysis revealed that they share similar structure with 16:0 and 18:0 PAF. Furthermore, other lipids extracted from Z. mobilis were found to exhibit a potent anti-PAF activity. Enzyme activities indicative of key PAF biosynthetic enzymes, such as dithiothreitol-insensitive cholinephosphotransferase (PAF-CPT) and lyso-PAF acetyltransferase were detected in Z. mobilis homogenates. As for PAF degradation, activity similar to that of PAF acetylhydrolase was also discovered. Overall, the presence of PAF, PAF-specific inhibitors, and enzyme activities relating to PAF metabolism, suggests that PAF may play an intrinsic role in this biotechnological organism. Practical applications:Z. mobilis is a platform microorganism for bioethanol production and a potential source of high-value chemicals of interest to the food and healthcare industries. Further investigation of PAF's role is bound to affect applications involving this and other biotechnological organisms. The finding that Z. mobilis lipids exhibit potent anti-PAF activity opens up prospects for their identification, overproduction and pharmaceutical use. The presented HPLC method for lipid fractionation accomplishes a one-step separation of lipids from dense samples, which may be successfully employed to other lipid-rich sources such as blood.

Lysine acetylation is a well-established post-translational modification widely conserved and distributed in bacteria. Although multiple regulatory roles have been proved, little is known about its regulation. Here, we present evidence that the transcription of the Gcn5-like acetyltransferase YfiQ of Escherichia coli (proposed name: PatZ) is regulated by cAMP-CRP and its implications on acetate metabolism regulation. The acetate scavenging acetyl-CoA synthetase (Acs) is regulated at the transcriptional and post-translational levels. Post-translational regulation depends on a protein acetyltransferase (yfiQ) and an NAD(+) -dependent deacetylase (cobB). We have studied their expression under different environmental conditions. cobB is constitutively expressed from a promoter located upstream nagK. The expression of yfiQ occurs from its own promoter; it is upregulated in the stationary phase and in the presence of non-PTS carbon sources and is positively regulated by cAMP-CRP. Two putative CRP binding sites are necessary for its full activity. Gene deletion revealed that cobB is essential for growth on acetate, yfiQ deletion restoring growth of the cobB mutant. The fine tuning of metabolic enzymes results from the integration of multiple mechanisms, and redundant systems may exist. Despite the existence of divergent catabolite repression systems, this may be a conserved strategy common to both Gram-positive and -negative bacteria.

Scope: We examined the biological effect of gallic acid (GA) as a nuclear factor (NF)-κB acetyltransferase inhibitor on microglial-mediated β-amyloid neurotoxicity and restorative effects on the Aβ-induced cognitive dysfunction. Methods and results: The protective effects of GA on the survival of neuronal cells were assessed with an MTT assay and a co-culture system. For the co-culture experiments, both BV-2 and primary microglia cells were treated with GA prior to Aβ stimulation, and conditioned media were transferred to Neuro-2A cells. The mRNA and protein levels of inflammatory cytokines in both microglia and Neuro-2A cells were assessed with real-time polymerase chain reaction and western blotting. Inhibition of nuclear factor kappa B (NF-κB) acetylation with GA treatment resulted in reduced cytokine production in microglia cells and protection of neuronal cells from Aβ-induced neurotoxicity. Furthermore, we observed a restorative effect of GA on Aβ-induced cognitive dysfunction in mice with Y-maze and passive avoidance tests. Finally, we found that GA treatment efficiently blocked neuronal cell death by downregulating the expression of cytokines and the in vivo levels of NF-κB acetylation. Conclusion: These results suggest that selective inhibition of NF-κB acetylation by the histone acetyltransferase inhibitor GA is a possible therapeutic approach for alleviating the inflammatory progression of Alzheimer disease.

Cotranslational protein N-terminal modifications, including proteolytic maturation such as initiator methionine excision by methionine aminopeptidases and N-terminal blocking, occur universally. Protein alpha-N-acetylation, or the transfer of the acetyl moiety of acetyl-coenzyme A to nascent protein N-termini, catalysed by multisubunit N-terminal acetyltransferase complexes, generally takes place during protein translation. Nearly all protein modifications are known to influence different protein aspects such as folding, stability, activity and localization, and several studies have indicated similar functions for protein alpha-N-acetylation. However, until recently, protein alpha-N-acetylation remained poorly explored, mainly due to the absence of targeted proteomics technologies. The recent emergence of N-terminomics technologies that allow isolation of protein N-terminal peptides, together with proteogenomics efforts combining experimental and informational content have greatly boosted the field of alpha-N-acetylation. In this review, we report on such emerging technologies as well as on breakthroughs in our understanding of protein N-terminal biology.

In eukaryotes, lysine acetylation is a well-established posttranslational modification that has been implicated in virtually all aspects of eukaryotic physiology. Although homologs of the enzymes that catalyze protein acetylation are widely conserved and distributed amongst bacterial species, not much is known about the impact of protein acetylation on bacterial physiology. Here, we present evidence that the Gcn5-like acetyltransferase YfiQ and the sirtuin deacetylase CobB play crucial roles in the transcription regulation of the periplasmic stress-responsive promoter cpxP when cells of Escherichia coli grow in the presence of glucose, an environment that induces protein acetylation. Under this growth condition, several acetylation sites were detected on three of the RNA polymerase (RNAP) subunits: β, β’ and α. We focused on acetylations of the carboxy-terminal domain (CTD) of α because of its relative small size and its limited acetylation. We determined that K298 of α is acetylated in a glucose and YfiQ-dependent manner and that K298 is specifically required for glucose-induced cpxP transcription. Since the αCTD aids in promoter recognition by RNA polymerase, we propose its acetylation may influence bacterial physiology through effects on gene expression.

Sophorolipids from the non-pathogenic yeast Candida bombicola are applied commercially as biodegradable, eco-friendly surface active agents. These sophorolipids are produced by cultivation in presence of a hydrophobic carbon source and are always constituted of a mixture of structurally related molecules. For some applications however, certain structural variants perform better than others. Acetylation of the sophorolipid molecule is such a parameter that gains interest because of its influence on water solubility, foaming properties, and biological activity. Fully unacetylated sophorolipids therefore are interesting metabolites but cannot be produced in a pure way by conventional cultivation. Here we report the identification of the acetyltransferase gene AT, responsible for acetylation of de novo synthesized sophorolipids in Candida bombicola. By the creation of a Δat deletion mutant, we could create a yeast strain producing purely unacetylated sophorolipids with a yield of 5 ± 0.7 g/L using rapeseed oil as hydrophobic carbon source. In contrast to the chemical production of unacetylated sophorolipids used nowadays, the microbial production leads to mainly lactonic sophorolipids, in addition to minor amounts of acidic sophorolipids. Biotechnol. Bioeng. 2011;108: 2923–2931. © 2011 Wiley Periodicals, Inc.

How different cell types with the same genotype are formed and heritability maintained is a fundamental question in biology. We utilized white-opaque switching in Candida albicans as a system to study mechanisms of cell-type formation and maintenance. Each cell type has tractable characters, which are maintained over many cell divisions. Cell-type specification is under the control of interlocking transcriptional feedback loops, with Wor1 being the master regulator of the opaque cell type. Here we show that deletion of RTT109, encoding the acetyltransferase for histone H3K56, impairs stochastic and environmentally stimulated white-opaque switching. Ectopic expression of WOR1 mostly bypasses the requirement for RTT109, but opaque cells lacking RTT109 cannot be maintained. We have also discovered that nicotinamide induces opaque cell formation, and this activity of nicotinamide requires RTT109. Reducing the copy number of HST3, which encodes the H3K56 deacetylase, also leads to increased opaque formation. We further show that the Hst3 level is downregulated in the presence of genotoxins and ectopic expression of HST3 blocks genotoxin induced switching. This finding links genotoxin induced switching to Hst3 regulation. Together, these findings suggest RTT109 and HST3 genes play an important role in the regulation of white-opaque switching in C. albicans.

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. Proteins 2011; © 2011 Wiley-Liss, Inc.

Reporter gene assays are commonly used in applied toxicology to measure the transcription of genes involved in toxic responses. In these reporter gene assays, transgenic cells are used, which contain a promoter–operator region of a gene of interest fused to a reporter gene. The transcription of the gene of interest can be measured by the detection of the reporter protein. Chloramphenicol acetyl transferase (CAT) is frequently used as a reporter protein in mammalian reporter gene assays. Although CAT can be measured by different detection systems, like enzymatic and immune assays, most of these tests are expensive, time-consuming and labor-intensive. The excellent characteristics of phages, like their high affinity and specificity, their fast, cheap and animal-friendly manufacturing process with low batch-to-batch variations and their stability, make them appropriate as alternatives for antibodies in detection assays. Therefore, in this study single-chain variable fragment (scFv) phages were selected with affinity for CAT. Several scFv phages were selected that showed affinity towards CAT in a screening ELISA. Surface plasmon resonance analyses showed that the tested scFv phages have an affinity for CAT with a dissociation constant (Kd) around 1 µM. The selected scFv phages in this study could be used as capture elements in a highly sensitive sandwich ELISA to detect CAT concentration as low as 0.1 ng ml−1 or 4 pM. This low detection limit demonstrates the potential of the scFv phages as an alternative for capturing antibodies in a highly sensitive detection test to measure CAT concentrations in reporter gene assays. Copyright © 2011 John Wiley & Sons, Ltd.

Extracts of sediments from an area of concern in the Elbe river basins (Spittelwasser creek) were analyzed with the Ames-fluctuation test and in parallel with gas chromatography/mass spectrometry for compound identification. The standard test strains TA 98 and TA 100 showed mutagenicity mainly in medium-polar fractions of the sediment extracts. PAHs contribute to the overall mutagenic potential of the sample. Especially, cyclopenta[c,d]pyrene that was previously not defined as a priority hazardous substance has to be considered as well. The addition of metabolically competent test strains, which overexpress nitroreductase and acetyltransferase (e.g., YG1041 and YG1042) to the test battery, increased significantly the sensitivity of the Ames test for medium polar to polar genotoxins. The increased mutagenicity that was found in these bacterial strains indicates the presence of nitroarenes and/or aromatic amines. In fact, a number of heterocyclic and nitrogen-substituted aromatic compounds were identified in the sediments of the Spittelwasser creek of which methyl parathion, 1-naphthylamine, and N-phenyl-2-naphthylamine are mutagenic.

Mithramycin and chromomycin A(3) are two structurally related antitumour compounds, which differ in the glycosylation profiles and functional group substitutions of the sugars. Chromomycin contains two acetyl groups, which are incorporated during the biosynthesis by the acetyltransferase CmmA in Streptomyces griseus ssp. griseus. A bioconversion strategy using an engineered S. griseus strain generated seven novel acetylated mithramycins. The newly formed compounds were purified and characterized by MS and NMR. These new compounds differ from their parental compounds in the presence of one, two or three acetyl groups, attached at 3E, 4E and/or 4D positions. All new mithramycin analogues showed antitumour activity at micromolar of lower concentrations. Some of the compounds showed improved activities against glioblastoma or pancreas tumour cells. The CmmA acetyltransferase was located in the cell membrane and was shown to accept several acyl-CoA substrates. All these results highlight the potential of CmmA as a tool to create structural diversity in these antitumour compounds.