Structural Characterization of the Reaction and Substrate Specificity Mechanisms of Pathogenic Fungal Acetyl-CoA Synthetases.

Acetyl CoA synthetases (ACSs) are Acyl-CoA/NRPS/Luciferase (ANL) superfamily enzymes that couple acetate with CoA to generate acetyl CoA, a key component of central carbon metabolism in eukaryotes and prokaryotes. Normal mammalian cells are not dependent on ACSs, while tumor cells, fungi, and parasites rely on acetate as a precursor for acetyl CoA. Consequently, ACSs have emerged as a potential drug target. As part of a program to develop antifungal ACS inhibitors, we characterized fungal ACSs from five diverse human fungal pathogens using biochemical and structural studies. ACSs catalyze a two-step reaction involving adenylation of acetate followed by thioesterification with CoA. Our structural studies captured each step of these two half-reactions including the acetyl-adenylate intermediate of the first half-reaction in both the adenylation conformation and the thioesterification conformation and thus provide a detailed picture of the reaction mechanism. We also used a systematic series of increasingly larger alkyl adenosine esters as chemical probes to characterize the structural basis of the exquisite ACS specificity for acetate over larger carboxylic acid substrates. Consistent with previous biochemical and genetic data for other enzymes, structures of fungal ACSs with these probes bound show that a key tryptophan residue limits the size of the alkyl binding site and forces larger alkyl chains to adopt high energy conformers, disfavoring their efficient binding. Together, our analysis provides highly detailed structural models for both the reaction mechanism and substrate specificity that should be useful in designing selective inhibitors of eukaryotic ACSs as potential anticancer, antifungal, and antiparasitic drugs.

Mammalian acetate-dependent acetyl CoA synthetase 2 contains multiple protein destabilization and masking elements.

Besides contributing to anabolism, cellular metabolites serve as substrates or cofactors for enzymes and may also have signaling functions. Given these roles, multiple control mechanisms likely ensure fidelity of metabolite-generating enzymes. Acetate-dependent acetyl CoA synthetases (ACS) are de novo sources of acetyl CoA, a building block for fatty acids and a substrate for acetyltransferases. Eukaryotic acetate-dependent acetyl CoA synthetase 2 (Acss2) is predominantly cytosolic, but is also found in the nucleus following oxygen or glucose deprivation, or upon acetate exposure. Acss2-generated acetyl CoA is used in acetylation of Hypoxia-Inducible Factor 2 (HIF-2), a stress-responsive transcription factor. Mutation of a putative nuclear localization signal in endogenous Acss2 abrogates HIF-2 acetylation and signaling, but surprisingly also results in reduced Acss2 protein levels due to unmasking of two protein destabilization elements (PDE) in the Acss2 hinge region. In the current study, we identify up to four additional PDE in the Acss2 hinge region and determine that a previously identified PDE, the ABC domain, consists of two functional PDE. We show that the ABC domain and other PDE are likely masked by intramolecular interactions with other domains in the Acss2 hinge region. We also characterize mice with a prematurely truncated Acss2 that exposes a putative ABC domain PDE, which exhibits reduced Acss2 protein stability and impaired HIF-2 signaling. Finally, using primary mouse embryonic fibroblasts, we demonstrate that the reduced stability of select Acss2 mutant proteins is due to a shortened half-life, which is a result of enhanced degradation via a nonproteasome, nonautophagy pathway.

Screening of DNA-Encoded Small Molecule Libraries inside a Living Cell.

DNA-encoded small molecule libraries (DELs) have facilitated the discovery of novel modulators of many different therapeutic protein targets. We report the first successful screening of a multimillion membered DEL inside a living cell. We demonstrate a novel method using oocytes from the South African clawed frog Xenopus laevis. The large size of the oocytes of 1 µL, or 100 000 times bigger than a normal somatic cell, permits simple injection of DELs, thus resolving the fundamental problem of delivering DELs across cell membranes for in vivo screening. The target protein was expressed in the oocytes fused to a prey protein, to allow specific DNA labeling and hereby discriminate between DEL members binding to the target protein and the endogenous cell proteins. The 194 million member DEL was screened against three pharmaceutically relevant protein targets, p38α, ACSS2, and DOCK5. For all three targets multiple chemical clusters were identified. For p38α, validated hits with single digit nanomolar potencies were obtained. This work demonstrates a powerful new approach to DEL screening, which eliminates the need for highly purified active target protein and which performs the screening under physiological relevant conditions and thus is poised to increase the DEL amenable target space and reduce the attrition rates.

Insights into the Chemical Reactivity in Acetyl-CoA Synthase.

The biological synthesis of acetyl-coenzyme A (acetyl-CoA), catalyzed by acetyl-CoA synthase (ACS), is of biological significance and chemical interest acting as a source of energy and carbon. The catalyst contains an unusual hexa-metal cluster with two nickel ions and a [Fe4S4] cluster. DFT calculations have been performed to investigate the ACS reaction mechanism starting from three different oxidation states (+2, +1, and 0) of Nip, the nickel proximal to [Fe4S4]. The results indicate that the ACS reaction proceeds first through a methyl radical transfer from cobalamin (Cbl) to Nip randomly accompanying with the CO binding. After that, C-C bond formation occurs between the Nip-bound methyl and CO, forming Nip-acetyl. The substrate CoA-S- then binds to Nip, allowing C-S bond formation between the Nip-bound acetyl and CoA-S-. Methyl transfer is rate-limiting with a barrier of ∼14 kcal/mol, which does not depend on the presence or absence of CO. Both the Nip2+ and Nip1+ states are chemically capable of catalyzing the ACS reaction independent of the state (+2 or +1) of the [Fe4S4] cluster. The [Fe4S4] cluster is not found to affect the steps of methyl transfer and C-C bond formation but may be involved in the C-S bond formation depending on the detailed mechanism chosen. An ACS active site containing a Nip(0) state could not be obtained. Optimizations always led to a Nip1+ state coupled with [Fe4S4]1+. The calculations show a comparable activity for Nip1+/[Fe4S4]1+, Nip1+/[Fe4S4]2+, and Nip2+/[Fe4S4]2+. The results here give significant insights into the chemistry of the important ACS reaction.

Fragmentation of acetate-CoA ligase gives a clue to understand domain rearrangement history of NDP-forming acyl-CoA synthetase superfamily proteins.

NDP-forming type acyl-CoA synthetase superfamily proteins are known to have six essential subdomains (1, 2, 3, a, b, c) of which partition and order are varied, suggesting yet-to-be-defined subdomain rearrangement happened in its evolution. Comparison in physicochemical and biochemical characteristics between the recombinant proteins which we made from fragmented subdomains and wild-type protein, acetate-CoA ligase in a hyperthermophilic archaeon, consisting of two distinct subunits (α1-2-3 and ßa-b-c) provided a clue to the mystery of its molecular evolutionary passage. Although solubility and thermostability of each fragmented subdomain turned out to be lower than that of wild-type, mixture of the three synthetic subunits of α1-2, α3, and ßa-b-c had quaternary structure, thermostability, and enzymatic activity comparable to those of the wild-type. This suggests that substantial independence and mobility of subdomain 3 have enabled rearrangement of the subdomains; and thermostability of the subdomains has constrained the composition of the subunits.

A substitution mutation in a conserved domain of mammalian acetate-dependent acetyl CoA synthetase 2 results in destabilized protein and impaired HIF-2 signaling.

The response to environmental stresses by eukaryotic organisms includes activation of protective biological mechanisms, orchestrated in part by transcriptional regulators. The tri-member Hypoxia Inducible Factor (HIF) family of DNA-binding transcription factors include HIF-2, which is activated under conditions of oxygen or glucose deprivation. Although oxygen-dependent protein degradation is a key mechanism by which HIF-1 and HIF-2 activity is regulated, HIF-2 is also influenced substantially by the coupled action of acetylation and deacetylation. The acetylation/deacetylation process that HIF-2 undergoes employs a specific acetyltransferase and deacetylase. Likewise, the supply of the acetyl donor, acetyl CoA, used for HIF-2 acetylation originates from a specific acetyl CoA generator, acetate-dependent acetyl CoA synthetase 2 (Acss2). Although Acss2 is predominantly cytosolic, a subset of the Acss2 cellular pool is enriched in the nucleus following oxygen or glucose deprivation. Prevention of nuclear localization by a directed mutation in a putative nuclear localization signal in Acss2 abrogates HIF-2 acetylation and blunts HIF-2 dependent signaling as well as flank tumor growth for knockdown/rescue cancer cells expressing ectopic Acss2. In this study, we report generation of a novel mouse strain using CRISPR/Cas9 mutagenesis that express this mutant Acss2 allele in the mouse germline. The homozygous mutant mice have impaired induction of the canonical HIF-2 target gene erythropoietin and blunted recovery from acute anemia. Surprisingly, Acss2 protein levels are dramatically reduced in these mutant mice. Functional studies investigating the basis for this phenotype reveal multiple protein instability domains in the Acss2 carboxy terminus. The findings described herein may be of relevance in the regulation of native Acss2 protein as well as for humans carrying missense mutations in these domains.

Characterization of Microalgal Acetyl-CoA Synthetases with High Catalytic Efficiency Reveals Their Regulatory Mechanism and Lipid Engineering Potential.

Acetyl-CoA synthetase (ACS) plays a key role in microalgal lipid biosynthesis and acetyl-CoA industrial production. In the present study, two ACSs were cloned and characterized from the oleaginous microalga Chromochloris zofingiensis. In vitro kinetic analysis showed that the Km values of CzACS1 and CzACS2 for potassium acetate were 0.99 and 0.81 mM, respectively. Moreover, CzACS1 and CzACS2 had outstanding catalytic efficiencies (kcat/Km), which were 70.67 and 79.98 s-1 mM-1, respectively, and these values were higher than that of other reported ACSs. CzACS1 and CzACS2 exhibited differential expression patterns at the transcriptional level under various conditions. Screening a recombinant library of 52 transcription factors (TFs) constructed in the present study via yeast one-hybrid assay pointed to seven TFs with potential involvement in the regulation of the two ACS genes. Expression correlation analysis implied that GATA20 was likely an important regulator of CzACS2 and that ERF9 could regulate two CzACSs simultaneously.

Staphylococcus aureus modulates the activity of acetyl-Coenzyme A synthetase (Acs) by sirtuin-dependent reversible lysine acetylation.

Lysine acylation is a posttranslational modification used by cells of all domains of life to modulate cellular processes in response to metabolic stress. The paradigm for the role of lysine acylation in metabolism is the acetyl-coenzyme A synthetase (Acs) enzyme. In prokaryotic and eukaryotic cells alike, Acs activity is downregulated by acetylation and reactivated by deacetylation. Proteins belonging to the bacterial GCN5-related N-acetyltransferase (bGNAT) superfamily acetylate the epsilon amino group of an active site lysine, inactivating Acs. A deacetylase can remove the acetyl group, thereby restoring activity. Here we show the Acs from Staphylococcus aureus (SaAcs) activates acetate and weakly activates propionate, but does not activate >C3 organic acids or dicarboxylic acids (e.g. butyrate, malonate and succinate). SaAcs activity is regulated by AcuA (SaAcuA); a type-IV bGNAT. SaAcuA can acetylate or propionylate SaAcs reducing its activity by >90% and 95% respectively. SaAcuA also succinylated SaAcs, with this being the first documented case of a bacterial GNAT capable of succinylation. Inactive SaAcsAc was deacetylated (hence reactivated) by the NAD+ -dependent (class III) sirtuin protein deacetylase (hereafter SaCobB). In vivo and in vitro evidence show that SaAcuA and SaCobB modulate the level of SaAcs activity in S. aureus.

Altering the Substrate Specificity of Acetyl-CoA Synthetase by Rational Mutagenesis of the Carboxylate Binding Pocket.

Acetyl-CoA synthetase (ACS) is a member of a large superfamily of enzymes that display diverse substrate specificities, with a common mechanism of catalyzing the formation of a thioester bond between Coenzyme A and a carboxylic acid, while hydrolyzing ATP to AMP and pyrophosphate. As an activated form of acetate, acetyl-CoA is a key metabolic intermediate that links many metabolic processes, including the TCA cycle, amino acid metabolism, fatty acid metabolism and biosynthetic processes that generate many polyketides and some terpenes. We explored the structural basis of the specificity of ACS for only activating acetate, whereas other members of this superfamily utilize a broad range of other carboxylate substrates. By computationally modeling the structure of the Arabidopsis ACS and the Pseudomonas chlororaphis isobutyryl-CoA synthetase using the experimentally determined tertiary structures of homologous ACS enzymes as templates, we identified residues that potentially comprise the carboxylate binding pocket. These predictions were systematically tested by mutagenesis of four specific residues. The resulting rationally redesigned carboxylate binding pocket modified the size and chemo-physical properties of the carboxylate binding pocket. This redesign successfully switched a highly specific enzyme from using only acetate, to be equally specific for using longer linear (up to hexanoate) or branched chain (methylvalerate) carboxylate substrates. The significance of this achievement is that it sets a precedent for understanding the structure-function relationship of an enzyme without the need for an experimentally determined tertiary structure of that target enzyme, and rationally generates new biocatalysts for metabolic engineering of a broad range of metabolic processes.

Acetyl-coenzyme A synthetase gene ChAcs1 is essential for lipid metabolism, carbon utilization and virulence of the hemibiotrophic fungus Colletotrichum higginsianum.

Acetyl-coenzyme A (acetyl-CoA) is a key molecule that participates in many biochemical reactions in amino acid, protein, carbohydrate and lipid metabolism. Here, we genetically dissected the distinct roles of two acetyl-CoA synthetase genes, ChAcs1 and ChAcs2, in the regulation of fermentation, lipid metabolism and virulence of the hemibiotrophic fungus Colletotrichum higginsianum. ChAcs1 and ChAcs2 are both highly expressed during appressorial development and the formation of primary hyphae, and are constitutively expressed in the cytoplasm throughout development. We found that C. higginsianum strains without ChAcs1 were non-viable in the presence of most non-fermentable carbon sources, including acetate, ethanol and acetaldehyde. Deletion of ChAcs1 also led to a decrease in lipid content of mycelia and delayed lipid mobilization in conidia to developing appressoria, which suggested that ChAcs1 contributes to lipid metabolism in C. higginsianum. Furthermore, a ChAcs1 deletion mutant was defective in the switch to invasive growth, which may have been directly responsible for its reduced virulence. Transcriptomic analysis and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) revealed that ChAcs1 can affect the expression of genes involved in virulence and carbon metabolism, and that plant defence genes are up-regulated, all demonstrated during infection by a ChAcs1 deletion mutant. In contrast, deletion of ChAcs2 only conferred a slight delay in lipid mobilization, although it was highly expressed in infection stages. Our studies provide evidence for ChAcs1 as a key regulator governing lipid metabolism, carbon source utilization and virulence of this hemibiotrophic fungus.

Model Complexes for the Nip Site of Acetyl Coenzyme A Synthase/Carbon Monoxide (CO) Dehydrogenase: Structure, Electrochemistry, and CO Reactivity.

Aliphatic thiolato-S-bridged tri- and binuclear nickel(II) complexes have been synthesized and characterized as models for the Nip site of the A cluster of acetyl coenzyme A synthase (ACS)/carbon monooxide (CO) dehydrogenase. Reaction of the in situ formed N2Sthiol donor ligands with [Ni(H2O)6](ClO4)2 afforded the trinuclear complexes [Ni{(LMe(S))2Ni}2](ClO4)2·CH3CN (1·CH3CN) and [Ni{(LBr(S))2Ni}2](ClO4)2·5H2O (2·5H2O) following self-assembly. Complexes 1 and 2 react with [Ni(dppe)Cl2] and dppe [dppe = 1,2-bis(diphenylphosphino)ethane] to afford the binuclear [Ni(dppe)Ni(LMe(S))2](ClO4)2·2H2O (3·2H2O) and [Ni(dppe)Ni(LBr(S))2](ClO4)2·0.75O(C2H5)2 [4·0.75O(C2H5)2], respectively. The X-ray crystal structures of 1-4 revealed a central NiIIS4 moiety in 1 and 2 and a NiIIP2S2 moiety in 3 and 4; both moieties have a square-planar environment around Ni and may mimic the properties of the Nip site of ACS. The electrochemical reduction of both terminal NiII ions of 1 and 2 occurs simultaneously, which is further confirmed by the isolation of [Ni{(LMe(S))2Ni(NO)}2](ClO4)2 (5) and [Ni{(LBr(S))2Ni(NO)}2](ClO4)2 (6) following reductive nitrosylation of 1 and 2. Complexes 5 and 6 exhibit νNO at 1773 and 1789 cm-1, respectively. In the presence of O2, both 5 and 6 transform to nitrite-bound monomers [(LMe(S-S))Ni(NO2)](ClO4) (7) and [(LBr(S-S))Ni(NO2)](ClO4)2 (8). The nature of the ligand modification is evident from the X-ray crystal structure of 7. To understand the origin of multiple reductive responses of 1-4, complex [(LMe(SMe))2Ni](ClO4)2 (9) is considered. The central NiS4 part of 1 is labile like the Nip site of ACS and can be replaced by phenanthroline. The treatment of CO to reduce 3 generates a 3red-(CO)2 species, as confirmed by Fourier transform infrared (νCO = 1997 and 2068 cm-1) and electron paramagnetic resonance ( g1 = 2.18, g2 = 2.13, g3 = 1.95, and AP = 30-80 G) spectroscopy. The CO binding to NiI of 3red is relevant to the ACS activity.

Toxoplasma gondii acetyl-CoA synthetase is involved in fatty acid elongation (of long fatty acid chains) during tachyzoite life stages.

Apicomplexan parasites are pathogens responsible for major human diseases such as toxoplasmosis caused by Toxoplasma gondii and malaria caused by Plasmodium spp. Throughout their intracellular division cycle, the parasites require vast and specific amounts of lipids to divide and survive. This demand for lipids relies on a fine balance between de novo synthesized lipids and scavenged lipids from the host. Acetyl-CoA is a major and central precursor for many metabolic pathways, especially for lipid biosynthesis. T. gondii possesses a single cytosolic acetyl-CoA synthetase (TgACS). Its role in the parasite lipid synthesis is unclear. Here, we generated an inducible TgACS KO parasite line and confirmed the cytosolic localization of the protein. We conducted 13C-stable isotope labeling combined with mass spectrometry-based lipidomic analyses to unravel its putative role in the parasite lipid synthesis pathway. We show that its disruption has a minor effect on the global FA composition due to the metabolic changes induced to compensate for its loss. However, we could demonstrate that TgACS is involved in providing acetyl-CoA for the essential fatty elongation pathway to generate FAs used for membrane biogenesis. This work provides novel metabolic insight to decipher the complex lipid synthesis in T. gondii.

Multielectron Chemistry within a Model Nickel Metalloprotein: Mechanistic Implications for Acetyl-CoA Synthase.

The acetyl coenzyme A synthase (ACS) enzyme plays a central role in the metabolism of anaerobic bacteria and archaea, catalyzing the reversible synthesis of acetyl-CoA from CO and a methyl group through a series of nickel-based organometallic intermediates. Owing to the extreme complexity of the native enzyme systems, the mechanism by which this catalysis occurs remains poorly understood. In this work, we have developed a protein-based model for the NiP center of acetyl coenzyme A synthase using a nickel-substituted azurin protein (NiAz). NiAz is the first model nickel protein system capable of accessing three (NiI/NiII/NiIII) distinct oxidation states within a physiological potential range in aqueous solution, a critical feature for achieving organometallic ACS activity, and binds CO and -CH3 groups with biologically relevant affinity. Characterization of the NiI-CO species through spectroscopic and computational techniques reveals fundamentally similar features between the model NiAz system and the native ACS enzyme, highlighting the potential for related reactivity in this model protein. This work provides insight into the enzymatic process, with implications toward engineering biological catalysts for organometallic processes.

Ligand binding at the A-cluster in full-length or truncated acetyl-CoA synthase studied by X-ray absorption spectroscopy.

Bacteria integrate CO2 reduction and acetyl coenzyme-A (CoA) synthesis in the Wood-Ljungdal pathway. The acetyl-CoA synthase (ACS) active site is a [4Fe4S]-[NiNi] complex (A-cluster). The dinickel site structure (with proximal, p, and distal, d, ions) was studied by X-ray absorption spectroscopy in ACS variants comprising all three protein domains or only the C-terminal domain with the A-cluster. Both variants showed two square-planar Ni(II) sites and an OH- bound at Ni(II)p in oxidized enzyme and a H2O at Ni(I)p in reduced enzyme; a Ni(I)p-CO species was induced by CO incubation and a Ni(II)-CH3- species with an additional water ligand by a methyl group donor. These findings render a direct effect of the N-terminal and middle domains on the A-cluster structure unlikely.

Structure of NDP-forming Acetyl-CoA synthetase ACD1 reveals a large rearrangement for phosphoryl transfer.

The NDP-forming acyl-CoA synthetases (ACDs) catalyze the conversion of various CoA thioesters to the corresponding acids, conserving their chemical energy in form of ATP. The ACDs are the major energy-conserving enzymes in sugar and peptide fermentation of hyperthermophilic archaea. They are considered to be primordial enzymes of ATP synthesis in the early evolution of life. We present the first crystal structures, to our knowledge, of an ACD from the hyperthermophilic archaeon Candidatus Korachaeum cryptofilum. These structures reveal a unique arrangement of the ACD subunits alpha and beta within an α2ß2-heterotetrameric complex. This arrangement significantly differs from other members of the superfamily. To transmit an activated phosphoryl moiety from the Ac-CoA binding site (within the alpha subunit) to the NDP-binding site (within the beta subunit), a distance of 51 Å has to be bridged. This transmission requires a larger rearrangement within the protein complex involving a 21-aa-long phosphohistidine-containing segment of the alpha subunit. Spatial restraints of the interaction of this segment with the beta subunit explain the necessity for a second highly conserved His residue within the beta subunit. The data support the proposed four-step reaction mechanism of ACDs, coupling acyl-CoA thioesters with ATP synthesis. Furthermore, the determined crystal structure of the complex with bound Ac-CoA allows first insight, to our knowledge, into the determinants for acyl-CoA substrate specificity. The composition and size of loops protruding into the binding pocket of acyl-CoA are determined by the individual arrangement of the characteristic subdomains.

Two acetyl-CoA synthetase isoenzymes are encoded by distinct genes in marine yeast Rhodosporidium diobovatum.

OBJECTIVES: Two genes encoding two acetyl-CoA synthetase (ACS) isoenzymes have been identified in the marine yeast Rhodosporidium diobovatum MCCC 2A00023. RESULTS: ACS1 encoded a polypeptide with a sequence of 578 amino acid residues, a predicted molecular weight of 63.73 kDa, and pI of 8.14, while the ACS2 encoded a polypeptide containing 676 amino acid residues with a deduced molecular mass of 75.61 kDa and a pI of 5.95. Biological activity of Acs1p and Acs2p was confirmed by heterologous expression in Escherichia coli. A 1.5-kb DNA fragment of the ACS1 gene and a 2.7-kb DNA fragment of the ACS2 gene were deleted using the RNA guide CRISPR-Cas9 system. The strain lacking ACS1 was unable to grow on acetate and ethanol media, while the ACS2 deletant was unable to grow on glucose medium. ACS1-ACS2 double mutants of R. diobovatum were non-viable. CONCLUSIONS: ACS isoenzymes are essential to the yeast metabolism, and other sources of ACSs cannot compensate for the lack of ACSs encoded by the two genes.

Regulation of a Protein Acetyltransferase in Myxococcus xanthus by the Coenzyme NADP.

UNLABELLED: NADP(+) is a vital cofactor involved in a wide variety of activities, such as redox potential and cell death. Here, we show that NADP(+) negatively regulates an acetyltransferase from Myxococcus xanthus, Mxan_3215 (MxKat), at physiologic concentrations. MxKat possesses an NAD(P)-binding domain fused to the Gcn5-type N-acetyltransferase (GNAT) domain. We used isothermal titration calorimetry (ITC) and a coupled enzyme assay to show that NADP(+) bound to MxKat and that the binding had strong effects on enzyme activity. The Gly11 residue of MxKat was confirmed to play an important role in NADP(+) binding using site-directed mutagenesis and circular dichroism spectrometry. In addition, using mass spectrometry, site-directed mutagenesis, and a coupling enzymatic assay, we demonstrated that MxKat acetylates acetyl coenzyme A (acetyl-CoA) synthetase (Mxan_2570) at Lys622 in response to changes in NADP(+) concentration. Collectively, our results uncovered a mechanism of protein acetyltransferase regulation by the coenzyme NADP(+) at physiological concentrations, suggesting a novel signaling pathway for the regulation of cellular protein acetylation. IMPORTANCE: Microorganisms have developed various protein posttranslational modifications (PTMs), which enable cells to respond quickly to changes in the intracellular and extracellular milieus. This work provides the first biochemical characterization of a protein acetyltransferase (MxKat) that contains a fusion between a GNAT domain and NADP(+)-binding domain with Rossmann folds, and it demonstrates a novel signaling pathway for regulating cellular protein acetylation in M. xanthus. We found that NADP(+) specifically binds to the Rossmann fold of MxKat and negatively regulates its acetyltransferase activity. This finding provides novel insight for connecting cellular metabolic status (NADP(+) metabolism) with levels of protein acetylation, and it extends our understanding of the regulatory mechanisms underlying PTMs.

Potential Role of Acetyl-CoA Synthetase (acs) and Malate Dehydrogenase (mae) in the Evolution of the Acetate Switch in Bacteria and Archaea.

Although many Archaea have AMP-Acs (acetyl-coenzyme A synthetase) and ADP-Acs, the extant methanogenic genus Methanosarcina is the only identified Archaeal genus that can utilize acetate via acetate kinase (Ack) and phosphotransacetylase (Pta). Despite the importance of ack as the potential urkinase in the ASKHA phosphotransferase superfamily, an origin hypothesis does not exist for the acetate kinase in Bacteria, Archaea, or Eukarya. Here we demonstrate that Archaeal AMP-Acs and ADP-Acs contain paralogous ATPase motifs previously identified in Ack, which demonstrate a novel relation between these proteins in Archaea. The identification of ATPase motif conservation and resulting structural features in AMP- and ADP-acetyl-CoA synthetase proteins in this study expand the ASKHA superfamily to include acetyl-CoA synthetase. Additional phylogenetic analysis showed that Pta and MaeB sequences had a common ancestor, and that the Pta lineage within the halophilc archaea was an ancestral lineage. These results suggested that divergence of a duplicated maeB within an ancient halophilic, archaeal lineage formed a putative pta ancestor. These results provide a potential scenario for the establishment of the Ack/Pta pathway and provide novel insight into the evolution of acetate metabolism for all three domains of life.

Influence of key amino acid mutation on the active site structure and on folding in acetyl-CoA synthase: a theoretical perspective.

Ad hoc quantum chemical modeling of the acetyl-CoA synthase local structure and folding allowed us to identify an unprecedented coordination mode of histidine sidechain to protein-embedded metal ions.

Lipid metabolism in response to individual short chain fatty acids during mixotrophic mode of microalgal cultivation: Influence on biodiesel saturation and protein profile.

Critical influence of different short chain fatty acids as organic carbon source, during growth (GP) and nutrient stress lipogenic phase (NSLP) was investigated on biomass and lipid productivity, in mixotrophic fed-batch microalgae cultivation. Nutrient deprivation induced physiological stress stimulated highest lipid productivity with acetate (total/neutral lipids, 35/17) with saturation index of 80.53% by the end of NSLP followed by butyrate (12/7%; 78%). Biomass growth followed the order of acetate (2.23 g/l) >butyrate (0.99 g/l) >propionate (0.77 g/l). VFA removal (as COD) was maximum with acetate (87%) followed by butyrate (55.09%) and propionate (10.60%). Palmitic acid was the most dominant fatty acid found in the fatty acid composition of all variants and butyrate fed system yielded a maximum of 44% palmitic acid. Protein profiling illustrated prominence of acetyl CoA-synthetase activity in acetate system. Thus, fatty acids provide a promising alternative feedstock for biodiesel production with integrated microalgae-biorefinery.