Molecules Of acetyl-CoA Research Articles

Production of succinate with two CO2 fixation reactions from fatty acids in Cupriavidus necator H16

BackgroundBiotransformation of CO2 into high-value-added carbon-based products is a promising process for reducing greenhouse gas emissions. To realize the green transformation of CO2, we use fatty acids as carbon source to drive CO2 fixation to produce succinate through a portion of the 3-hydroxypropionate (3HP) cycle in Cupriavidus necator H16.ResultsThis work can achieve the production of a single succinate molecule from one acetyl-CoA molecule and two CO2 molecules. It was verified using an isotope labeling experiment utilizing NaH13CO3. This implies that 50% of the carbon atoms present in succinate are derived from CO2, resulting in a twofold increase in efficiency compared to prior methods of succinate biosynthesis that relied on the carboxylation of phosphoenolpyruvate or pyruvate. Meanwhile, using fatty acid as a carbon source has a higher theoretical yield than other feedstocks and also avoids carbon loss during acetyl-CoA and succinate production. To further optimize succinate production, different approaches including the optimization of ATP and NADPH supply, optimization of metabolic burden, and optimization of carbon sources were used. The resulting strain was capable of producing succinate to a level of 3.6 g/L, an increase of 159% from the starting strain.ConclusionsThis investigation established a new method for the production of succinate by the implementation of two CO2 fixation reactions and demonstrated the feasibility of ATP, NADPH, and metabolic burden regulation strategies in biological carbon fixation.

Role of acetyl-CoA acetyltransferase 1 expression in the molecular mechanism of adenomyosis.

Adenomyosis is a benign uterine illness characterized by endometrial gland and stromal invasion into the myometrium. Acetyl-CoA acetyltransferase 1 (ACAT1) is an enzyme localized in mitochondria that is involved in ketogenesis and ketolysis processes by reversibly catalyzing the formation of acetoacetyl-CoA from two acetyl-CoA molecules. The current study investigated the expression of the ACAT1 molecule in tissue samples of patients diagnosed with adenomyosis and healthy endometrial tissues. It is aimed to determine the differences in ACAT1 gene expression and in this way to discover the first information about the role of ACAT1 in the development and molecular mechanism of adenomyosis. In the current retrospective study, formalin-fixed paraffin-embedded archival tissues were employed. A total of 76 patient samples were included in the study. Of these samples, 28 are adenomyotic tissue (Group I), 30 are eutopic endometrial tissue (Group II), and 18 are the Control Group. In these groups, the expression levels of the ACAT1 gene were determined by the reverse transcription-polymerase chain reaction method. When the expression results of the ACAT1 gene were evaluated, statistically significant differences were found between the groups (p<0.05). There was a difference between Group I-Group II and Group I-Control Group regarding the ACAT1 gene. No statistically significant change was observed between Group II and Control Group. It is a remarkable finding that the expression of ACAT1 in adenomyosis tissue is decreased compared with both eutopic endometrium and control groups tissues. The results suggest that ACAT1 may be associated with the molecular pathogenesis of adenomyosis.

Allosteric Site at the Biotin Carboxylase Dimer Interface Mediates Activation and Inhibition in Staphylococcus aureus Pyruvate Carboxylase.

Allosteric regulation of the essential anaplerotic enzyme, pyruvate carboxylase (PC), is vital for metabolic homeostasis. PC catalyzes the bicarbonate- and ATP-dependent carboxylation of pyruvate to form oxaloacetate. Dysregulation of PC activity can impact glucose and redox metabolism, which contributes to the pathogenicity of many diseases. To maintain homeostasis, PC is allosterically activated by acetyl-CoA and allosterically inhibited by l-aspartate. In this study, we further characterize the molecular basis of allosteric regulation in Staphylococcus aureus PC (SaPC) using slowly/nonhydrolyzable dethia analogues of acetyl-CoA and site-directed mutagenesis of residues at the biotin carboxylase homodimer interface. The dethia analogues fully activate SaPC but demonstrate significantly reduced binding affinities relative to acetyl-CoA. Residues Arg21, Lys46, and Glu418 of SaPC are located at the biotin carboxylase dimer interface and play a critical role in both allosteric activation and inhibition. A structure of R21A SaPC in complex with acetyl-CoA reveals an intact molecule of acetyl-CoA bound at the allosteric site, offering new molecular insights into the acetyl-CoA binding site. This study demonstrates that the biotin carboxylase domain dimer interface is a critical allosteric site in PC, serving as a convergence point for allosteric activation by acetyl-CoA and inhibition by l-aspartate.

Functional evaluation of non-oxidative glycolysis in Escherichia coli in the stationary phase under microaerobic conditions

In microbial bioproduction, CO2 emissions via pyruvate dehydrogenase in the Embden-Meyerhof pathway, which converts glucose to acetyl-CoA, is one of the challenges for enhancing carbon yield. The synthetic non-oxidative glycolysis (NOG) pathway transforms glucose into three acetyl-CoA molecules without CO2 emission, making it an attractive module for metabolic engineering. Because the NOG pathway generates no ATP and NADH, it is expected to use a resting cell reaction. Therefore, it is important to characterize the feasibility of the NOG pathway during stationary phase. Here, we experimentally evaluated the invivo metabolic flow of the NOG pathway in Escherichia coli. An engineered strain was constructed by introducing phosphoketolase from Bifidobacterium adolescentis into E.coli and by deleting competitive reactions. When the strain was cultured in magnesium-starved medium under microaerobic conditions, the carbon yield of acetate, an end-product of the NOG pathway, was six times higher than that of the control strain harboring an empty vector. Based on the mass balance constraints, the NOG flux was estimated to be between 2.89 and 4.64mmolg-1h-1, suggesting that the engineered cells can convert glucose through the NOG pathway with enough activity for bioconversion. Furthermore, to expand the application potential of NOG pathway-implemented strains, the theoretical maximum yields of various useful compounds were calculated using flux balance analysis. This suggests that the theoretical maximum yields of not only acetate but also lactam compounds can be increased by introducing the NOG pathway. This information will help in future applications of the NOG pathway.

Functional genomics-enabled characterization of CYP81B140 and CYP81B141 from Plumbago zeylanica L. substantiates their involvement in plumbagin biosynthesis.

Novel cytochrome P450s, CYP81B140 and CYP81B141 from Plumbago zeylanica were functionally characterized to understand their involvement in polyketide plumbagin biosynthesis. Further, we propose 3-methyl-1-8-naphthalenediol and isoshinanolone as intermediates for plumbagin biosynthesis. Plumbago zeylanica L. (P. zeylanica) is a medicinally important plant belonging to the family Plumbaginaceae. It comprises the most abundant naphthoquinone plumbagin having anti-cancer activity. Only the polyketide synthase (PKS) enzyme has been identified from the biosynthetic pathway which catalyzes iterative condensation of acetyl-CoA and malonyl-CoA molecules. The plumbagin biosynthesis involves hydroxylation, oxidation, hydration and dehydration of intermediate compounds which are expected to be catalyzed by cytochrome P450s (CYPs). To identify the CYPs, co-expression analysis was carried out using PKS as a candidate gene. Out of the eight identified CYPs, CYP81B140 and CYP81B141 have similar expression with PKS and belong to the CYP81 family. Phylogenetic analysis suggested that CYP81B140 and CYP81B141 cluster with CYPs from CYP81B, CYP81D, CYP81E and CYP81AA subfamilies which are known to be involved in the hydroxylation and oxidation reactions. Moreover, artificial microRNA-mediated transient individual silencing and co-silencing of CYP81B140 and CYP81B141 significantly reduced plumbagin and increased the 3-methyl-1-8-naphthalenediol and isoshinanolone content. Based on metabolite analysis, we proposed that 3-methyl-1-8-naphthalenediol and isoshinanolone function as intermediates for plumbagin biosynthesis. Transient silencing, over-expression and docking analysis revealed that CYP81B140 is involved in C-1 oxidation, C-4 hydroxylation and [C2-C3] hydration of 3-methyl-1-8-naphthalenediol to form isoshinanolone, whereas CYP81B141 is catalyzing [C2-C3] dehydration and C-4 oxidation of isoshinanolone to form plumbagin. Our results indicated that both CYP81B140 and CYP81B141 are promiscuous and necessary for plumbagin biosynthesis. This is the first report of identification and functional characterization of P. zeylanica-specific CYPs involved in plumbagin biosynthetic pathway and in general hexaketide synthesis in plants.

(S)-3-Hydroxybutyryl-CoA Dehydrogenase From the Autotrophic 3-Hydroxypropionate/4-Hydroxybutyrate Cycle in Nitrosopumilus maritimus.

Ammonia-oxidizing archaea of the phylum Thaumarchaeota are among the most abundant organisms that exert primary control of oceanic and soil nitrification and are responsible for a large part of dark ocean primary production. They assimilate inorganic carbon via an energetically efficient version of the 3-hydroxypropionate/4-hydroxybutyrate cycle. In this cycle, acetyl-CoA is carboxylated to succinyl-CoA, which is then converted to two acetyl-CoA molecules with 4-hydroxybutyrate as the key intermediate. This conversion includes the (S)-3-hydroxybutyryl-CoA dehydrogenase reaction. Here, we heterologously produced the protein Nmar_1028 catalyzing this reaction in thaumarchaeon Nitrosopumilus maritimus, characterized it biochemically and performed its phylogenetic analysis. This NAD-dependent dehydrogenase is highly active with its substrate, (S)-3-hydroxybutyryl-CoA, and its low Km value suggests that the protein is adapted to the functioning in the 3-hydroxypropionate/4-hydroxybutyrate cycle. Nmar_1028 is homologous to the dehydrogenase domain of crotonyl-CoA hydratase/(S)-3-hydroxybutyryl-CoA dehydrogenase that is present in many Archaea. Apparently, the loss of the dehydratase domain of the fusion protein in the course of evolution was accompanied by lateral gene transfer of 3-hydroxypropionyl-CoA dehydratase/crotonyl-CoA hydratase from Bacteria. Although (S)-3-hydroxybutyryl-CoA dehydrogenase studied here is neither unique nor characteristic for the HP/HB cycle, Nmar_1028 appears to be the only (S)-3-hydroxybutyryl-CoA dehydrogenase in N. maritimus and is thus essential for the functioning of the 3-hydroxypropionate/4-hydroxybutyrate cycle and for the biology of this important marine archaeon.

In Vivo Estimation of Ketogenesis Using Metabolic Flux Analysis-Technical Aspects and Model Interpretation.

Ketogenesis occurs in liver mitochondria where acetyl-CoA molecules, derived from lipid oxidation, are condensed into acetoacetate (AcAc) and reduced to β-hydroxybutyrate (BHB). During carbohydrate scarcity, these two ketones are released into circulation at high rates and used as oxidative fuels in peripheral tissues. Despite their physiological relevance and emerging roles in a variety of diseases, endogenous ketone production is rarely measured in vivo using tracer approaches. Accurate determination of this flux requires a two-pool model, simultaneous BHB and AcAc tracers, and special consideration for the stability of the AcAc tracer and analyte. We describe the implementation of a two-pool model using a metabolic flux analysis (MFA) approach that simultaneously regresses liquid chromatography-tandem mass spectrometry (LC-MS/MS) ketone isotopologues and tracer infusion rates. Additionally, 1H NMR real-time reaction monitoring was used to evaluate AcAc tracer and analyte stability during infusion and sample analysis, which were critical for accurate flux calculations. The approach quantifies AcAc and BHB pool sizes and their rates of appearance, disposal, and exchange. Regression analysis provides confidence intervals and detects potential errors in experimental data. Complications for the physiological interpretation of individual ketone fluxes are discussed.

Overcoming Energetic Barriers in Acetogenic C1 Conversion.

Currently one of the biggest challenges for society is to combat global warming. A solution to this global threat is the implementation of a CO2-based bioeconomy and a H2-based bioenergy economy. Anaerobic lithotrophic bacteria such as the acetogenic bacteria are key players in the global carbon and H2 cycle and thus prime candidates as driving forces in a H2- and CO2-bioeconomy. Naturally, they convert two molecules of CO2 via the Wood-Ljungdahl pathway (WLP) to one molecule of acetyl-CoA which can be converted to different C2-products (acetate or ethanol) or elongated to C4 (butyrate) or C5-products (caproate). Since there is no net ATP generation from acetate formation, an electron-transport phosphorylation (ETP) module is hooked up to the WLP. ETP provides the cell with additional ATP, but the ATP gain is very low, only a fraction of an ATP per mol of acetate. Since acetogens live at the thermodynamic edge of life, metabolic engineering to obtain high-value products is currently limited by the low energy status of the cells that allows for the production of only a few compounds with rather low specificity. To set the stage for acetogens as production platforms for a wide range of bioproducts from CO2, the energetic barriers have to be overcome. This review summarizes the pathway, the energetics of the pathway and describes ways to overcome energetic barriers in acetogenic C1 conversion.

Crystal structure of an acetyl-CoA acetyltransferase from PHB producing bacterium Bacillus cereus ATCC 14579

Bacillus cereus ATCC 14579 is a known polyhydroxybutyrate (PHB)-producing microorganism that possesses genes associated with PHB synthesis such as PhaA, PhaB, and PHA synthases. PhaA (i.e., thiolase) is the first enzyme in the PHA biosynthetic pathway, which catalyze the condensation of two acetyl-CoA molecules to acetoacetyl-CoA. Our study elucidated the crystal structure of PhaA in Bacillus cereus ATCC 14579 (BcTHL) in its apo- and CoA-bound forms. BcTHL adopts a type II biosynthetic thiolase structure by forming a tetramer. The crystal structure of CoA-complexed BcTHL revealed that the substrate binding site of BcTHL is constituted by different residues compared with other known thiolases. Our study also revealed that Arg221, a residue involved in ADP binding, undergoes a positional conformational change upon the binding of the CoA molecule.

Targeting mitochondrial metabolite transporters in Penicillium expansum for reducing patulin production

There is an increasing need of alternative treatments to control fungal infection and consequent mycotoxin accumulation in harvested fruits and vegetables. Indeed, only few biological targets of antifungal agents have been characterized and can be used for limiting fungal spread from decayed fruits/vegetables to surrounding healthy ones during storage. On this concern, a promising target of new antifungal treatments may be represented by mitochondrial proteins due to some species-specific functions played by mitochondria in fungal morphogenesis, drug resistance and virulence.One of the most studied mycotoxins is patulin produced by several species of Penicillium and Aspergillus genera. Patulin is toxic to many biological systems including bacteria, higher plants and animalia. Although precise biochemical mechanisms of patulin toxicity in humans are not completely clarified, its high presence in fresh and processed apple fruits and other apple-based products makes necessary developing a strategy for limiting its presence/accumulation. Patulin biosynthetic pathway consists of an enzymatic cascade, whose first step is represented by the synthesis of 6-methylsalicylic acid, obtained from the condensation of one acetyl-CoA molecule with three malonyl-CoA molecules. The most abundant acetyl-CoA precursor is represented by citrate produced by mitochondria. In the present investigation we report about the possibility to control patulin production through the inhibition of mitochondrial/peroxisome transporters involved in the export of acetyl-CoA precursors from mitochondria and/or peroxisomes, with specific reference to the predicted P. expansum mitochondrial Ctp1p, DTC, Sfc1p, Oac1p and peroxisomal PXN carriers.

Enzymes Catalyzing Crotonyl-CoA Conversion to Acetoacetyl-CoA During the Autotrophic CO2 Fixation in Metallosphaera sedula.

Autotrophic Crenarchaeota use two different cycles for carbon dioxide fixation. Members of the Sulfolobales use the 3-hydroxypropionate/4-hydroxybutyrate (HP/HB) cycle, whereas Desulfurococcales and Thermoproteales use the dicarboxylate/4-hydroxybutyrate cycle. While these two cycles differ in the carboxylation reactions resulting in the conversion of acetyl-CoA + 2 CO2 to succinyl-CoA, they have a common regeneration part in which succinyl-CoA is reconverted to two acetyl-CoA molecules. This common part includes crotonyl-CoA conversion to acetoacetyl-CoA, which has unequivocally been shown in Ignicoccus hospitalis (Desulfurococcales) and Pyrobaculum neutrophilus (Thermoproteales) to be catalyzed by a bifunctional crotonase/3-hydroxybutyryl-CoA dehydrogenase. It is a fusion protein consisting of an enoyl-CoA hydratase and a dehydrogenase domain. As the homologous bifunctional protein is present in Sulfolobales as well, its common functioning in the conversion of crotonyl-CoA to acetoacetyl-CoA was proposed. Here we show that a model autotrophic member of Sulfolobales, Metallosphaera sedula, possesses in addition to the bifunctional protein (Msed_0399) several separate genes coding for crotonyl-CoA hydratase and (S)-3-hydroxybutyryl-CoA dehydrogenase. Their genes were previously shown to be transcribed under autotrophic and mixotrophic conditions. The dehydrogenase Msed_1423 (and not the bifunctional protein Msed_0399) appears to be the main enzyme catalyzing the (S)-3-hydroxybutyryl-CoA dehydrogenase reaction. Homologs of this dehydrogenase are the only (S)-3-hydroxybutyryl-CoA dehydrogenases present in all autotrophic Sulfolobales, strengthening this conclusion. Two uncharacterized crotonase homologs present in M. sedula genome (Msed_0336 and Msed_0384) were heterologously produced and characterized. Both proteins were highly efficient crotonyl-CoA hydratases and may contribute (or be responsible) for the corresponding reaction in the HP/HB cycle in vivo.

Application of Stable Isotope Tracing to Elucidate Metabolic Dynamics During Yarrowia lipolytica α-Ionone Fermentation.

SummaryTargeted metabolite analysis in combination with 13C-tracing is a convenient strategy to determine pathway activity in biological systems; however, metabolite analysis is limited by challenges in separating and detecting pathway intermediates with current chromatographic methods. Here, a hydrophilic interaction chromatography tandem mass spectrometry approach was developed for improved metabolite separation, isotopologue analysis, and quantification. The physiological responses of a Yarrowia lipolytica strain engineered to produce ∼400 mg/L α-ionone and temporal changes in metabolism were quantified (e.g., mevalonate secretion, then uptake) indicating bottleneck shifts in the engineered pathway over the course of fermentation. Dynamic labeling results indicated limited tricarboxylic acid cycle label incorporation and, combined with a measurable ATP shortage during the high ionone production phase, suggested that electron transport and oxidative phosphorylation may limit energy supply and strain performance. The results provide insights into terpenoid pathway metabolic dynamics of non-model yeasts and offer guidelines for sensor development and modular engineering.

The role of the acyl-CoA thioesterase \u201cYciA\u201d in the production of (R)-3-hydroxybutyrate by recombinant Escherichia coli

Biotechnologically produced (R)-3-hydroxybutyrate is an interesting pre-cursor for antibiotics, vitamins, and other molecules benefitting from enantioselective production. An often-employed pathway for (R)-3-hydroxybutyrate production in recombinant E. coli consists of three-steps: (1) condensation of two acetyl-CoA molecules to acetoacetyl-CoA, (2) reduction of acetoacetyl-CoA to (R)-3-hydroxybutyrate-CoA, and (3) hydrolysis of (R)-3-hydroxybutyrate-CoA to (R)-3-hydroxybutyrate by thioesterase. Whereas for the first two steps, many proven heterologous candidate genes exist, the role of either endogenous or heterologous thioesterases is less defined. This study investigates the contribution of four native thioesterases (TesA, TesB, YciA, and FadM) to (R)-3-hydroxybutyrate production by engineered E. coli AF1000 containing a thiolase and reductase from Halomonas boliviensis. Deletion of yciA decreased the (R)-3-hydroxybutyrate yield by 43%, whereas deletion of tesB and fadM resulted in only minor decreases. Overexpression of yciA resulted in doubling of (R)-3-hydroxybutyrate titer, productivity, and yield in batch cultures. Together with overexpression of glucose-6-phosphate dehydrogenase, this resulted in a 2.7-fold increase in the final (R)-3-hydroxybutyrate concentration in batch cultivations and in a final (R)-3-hydroxybutyrate titer of 14.3 g L−1 in fed-batch cultures. The positive impact of yciA overexpression in this study, which is opposite to previous results where thioesterase was preceded by enzymes originating from different hosts or where (S)-3-hydroxybutyryl-CoA was the substrate, shows the importance of evaluating thioesterases within a specific pathway and in strains and cultivation conditions able to achieve significant product titers. While directly relevant for (R)-3-hydroxybutyrate production, these findings also contribute to pathway improvement or decreased by-product formation for other acyl-CoA-derived products.

Effect of precise control of flux ratio between the glycolytic pathways on mevalonate production in Escherichia coli.

Mevalonate is a useful metabolite synthesized from three molecules of acetyl-CoA, consuming two molecules of NADPH. Escherichia coli ( E. coli) catabolizes glucose to acetyl-CoA via several routes, such as the Embden-Meyerhof-Parnas (EMP) and the oxidative pentose phosphate (oxPP) pathways. Although the oxPP pathway supplies NADPH, it is disadvantageous in terms of acetyl-CoA supply, compared with the EMP pathway. In this study, the optimal flux ratio between the EMP and oxPP pathways on the mevalonate yield was investigated. Expression level of pgi was controlled by isopropyl β-D-1-thiogalactopyranoside (IPTG) inducible promoter in an engineered mevalonate-producing E. coli strain. The relationship between the flux ratio and mevalonate yield was evaluated by changing the flux ratio by varying IPTG concentration. At the stationary phase, the mevalonate yield was maximum at an EMP flux of 39.7%, and was increased by 25% compared with that with no flux control (EMP flux of 70.4%). The optimal flux ratio was consistent with the theoretical value based on the mass balance of NADPH. The flux ratio between EMP and oxPP pathways affects the synthesis fluxes of mevalonate and acetate from acetyl-CoA. Fine tuning of the flux ratio would be necessary to achieve an optimized production of metabolites that require NADPH.

Characterization of Mutant Sunflower Acetoacetyl CoA Thiolase

Sunflower (Helianthus annus L) cotyledons have two thiolase activities that have been identified previously. Thiolase II (acetoacetyl CoA thiolase) has specificity for the four‐carbon acetoacetyl CoA, resulting in the production of two Acetyl CoA molecules. Thiolase I (oxoacyl CoA thiolase) is active towards short, medium and long chain acyl CoAs. Crystal structures of thiolase from other organisms have identified conserved cysteines and a histidine present in the active site of the enzyme indicating that these residues probably play a key role in the reaction mechanism and other residues have been implicated in the stabilization of the enolate intermediate in the synthetic reaction. Site‐directed mutagenesis of the various amino acid residues was done and the effect of these mutations on the catalytic activity was measured.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Archaeal acetoacetyl-CoA thiolase/HMG-CoA synthase complex channels the intermediate via a fused CoA-binding site

Many reactions within a cell are thermodynamically unfavorable. To efficiently run some of those endergonic reactions, nature evolved intermediate-channeling enzyme complexes, in which the products of the first endergonic reactions are immediately consumed by the second exergonic reactions. Based on this concept, we studied how archaea overcome the unfavorable first reaction of isoprenoid biosynthesis-the condensation of two molecules of acetyl-CoA to acetoacetyl-CoA catalyzed by acetoacetyl-CoA thiolases (thiolases). We natively isolated an enzyme complex comprising the thiolase and 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase (HMGCS) from a fast-growing methanogenic archaeon, Methanothermococcus thermolithotrophicus HMGCS catalyzes the second reaction in the mevalonate pathway-the exergonic condensation of acetoacetyl-CoA and acetyl-CoA to HMG-CoA. The 380-kDa crystal structure revealed that both enzymes are held together by a third protein (DUF35) with so-far-unknown function. The active-site clefts of thiolase and HMGCS form a fused CoA-binding site, which allows for efficient coupling of the endergonic thiolase reaction with the exergonic HMGCS reaction. The tripartite complex is found in almost all archaeal genomes and in some bacterial ones. In addition, the DUF35 proteins are also important for polyhydroxyalkanoate (PHA) biosynthesis, most probably by functioning as a scaffold protein that connects thiolase with 3-ketoacyl-CoA reductase. This natural and highly conserved enzyme complex offers great potential to improve isoprenoid and PHA biosynthesis in biotechnologically relevant organisms.

Structure of Aspergillus fumigatus Cytosolic Thiolase: Trapped Tetrahedral Reaction Intermediates and Activation by Monovalent Cations

Cytosolic thiolase (CT) catalyzes the reversible Claisen condensation of two molecules of acetyl-CoA to produce acetoacetyl-CoA. The reaction cycle proceeds via a ping-pong mechanism involving an acetylated enzyme intermediate and two separate oxyanion holes which stabilize negatively charged reaction intermediates. This is the initial step in the synthesis of ergosterol in the prominent fungal pathogen Aspergillus fumigatus and is essential for the growth and survival of the organism. Here, we present crystal structures of A. fumigatus CT in liganded and apo forms and in a complex with different monovalent cations. Careful observation of the electron density at the active sites of two different afCT structures crystallized in the presence of acetyl-CoA shows that our crystals have trapped various stages of the thiolase catalytic cycle, including two tetrahedral reaction intermediates that have previously eluded structural characterization. Unexpectedly, we have also shown that afCT is activated by monova...

Succinyl-CoA:Mesaconate CoA-Transferase and Mesaconyl-CoA Hydratase, Enzymes of the Methylaspartate Cycle in Haloarcula hispanica.

Growth on acetate or other acetyl-CoA-generating substrates as a sole source of carbon requires an anaplerotic pathway for the conversion of acetyl-CoA into cellular building blocks. Haloarchaea (class Halobacteria) possess two different anaplerotic pathways, the classical glyoxylate cycle and the novel methylaspartate cycle. The methylaspartate cycle was discovered in Haloarcula spp. and operates in ∼40% of sequenced haloarchaea. In this cycle, condensation of one molecule of acetyl-CoA with oxaloacetate gives rise to citrate, which is further converted to 2-oxoglutarate and then to glutamate. The following glutamate rearrangement and deamination lead to mesaconate (methylfumarate) that needs to be activated to mesaconyl-C1-CoA and hydrated to β-methylmalyl-CoA. The cleavage of β-methylmalyl-CoA results in the formation of propionyl-CoA and glyoxylate. The carboxylation of propionyl-CoA and the condensation of glyoxylate with another acetyl-CoA molecule give rise to two C4-dicarboxylic acids, thus regenerating the initial acetyl-CoA acceptor and forming malate, its final product. Here we studied two enzymes of the methylaspartate cycle from Haloarcula hispanica, succinyl-CoA:mesaconate CoA-transferase (mesaconate CoA-transferase, Hah_1336) and mesaconyl-CoA hydratase (Hah_1340). Their genes were heterologously expressed in Haloferax volcanii, and the corresponding enzymes were purified and characterized. Mesaconate CoA-transferase was specific for its physiological substrates, mesaconate and succinyl-CoA, and produced only mesaconyl-C1-CoA and no mesaconyl-C4-CoA. Mesaconyl-CoA hydratase had a 3.5-fold bias for the physiological substrate, mesaconyl-C1-CoA, compared to mesaconyl-C4-CoA, and virtually no activity with other tested enoyl-CoA/3-hydroxyacyl-CoA compounds. Our results further prove the functioning of the methylaspartate cycle in haloarchaea and suggest that mesaconate CoA-transferase and mesaconyl-CoA hydratase can be regarded as characteristic enzymes of this cycle.

Malate Synthase and β-Methylmalyl Coenzyme A Lyase Reactions in the Methylaspartate Cycle in Haloarcula hispanica.

Acetate is one of the most important substances in natural environments. The activated form of acetate, acetyl coenzyme A (acetyl-CoA), is the high-energy intermediate at the crossroads of central metabolism: its oxidation generates energy for the cell, and about a third of all biosynthetic fluxes start directly from acetyl-CoA. Many organic compounds enter the central carbon metabolism via this key molecule. To sustain growth on acetyl-CoA-generating compounds, a dedicated assimilation (anaplerotic) pathway is required. The presence of an anaplerotic pathway is a prerequisite for growth in many environments, being important for environmentally, industrially, and clinically important microorganisms. Here we studied specific reactions of a recently discovered acetate assimilation pathway, the methylaspartate cycle, functioning in extremely halophilic archaea.

Crystal structure of a thiolase from Escherichia coli at 1.8 Å resolution.

Thiolases catalyze the Claisen condensation of two acetyl-CoA molecules to give acetoacetyl-CoA, as well as the reverse degradative reaction. Four genes coding for thiolases or thiolase-like proteins are found in the Escherichia coli genome. In this communication, the successful cloning, purification, crystallization and structure determination at 1.8 Å resolution of a homotetrameric E.coli thiolase are reported. The structure of E. coli thiolase co-crystallized with acetyl-CoA at 1.9 Å resolution is also reported. As observed in other tetrameric thiolases, the present E. coli thiolase is a dimer of two tight dimers and probably functions as a biodegradative enzyme. Comparison of the structure and biochemical properties of the E. coli enzyme with those of other well studied thiolases reveals certain novel features of this enzyme, such as the modification of a lysine in the dimeric interface, the possible oxidation of the catalytic Cys88 in the structure of the enzyme obtained in the presence of CoA and active-site hydration. The tetrameric enzyme also displays an interesting departure from exact 222 symmetry, which is probably related to the deformation of the tetramerization domain that stabilizes the oligomeric structure of the protein. The current study allows the identification of substrate-binding amino-acid residues and water networks at the active site and provides the structural framework required for understanding the biochemical properties as well as the physiological function of this E. coli thiolase.