Multienzyme mevalonate pathway bioreactor

The five-carbon metabolic intermediate isopentenyl diphosphate constitutes the basic building block for the biosynthesis of all isoprenoids in all forms of life. Two distinct pathways lead from amphibolic intermediates to isopentenyl diphosphate. The Gram-positive cocci and certain other pathogenic bacteria employ exclusively the mevalonate pathway, a set of six enzyme-catalyzed reactions that convert 3 mol of acetyl-CoA to 1 mol each of carbon dioxide and isopentenyl diphosphate. The survival of the Gram-positive cocci requires a fully functional set of mevalonate pathway enzymes. These enzymes therefore represent potential targets of inhibitors that might be employed as antibiotics directed against multidrug-resistant strains of certain bacterial pathogens. A rapid throughput, bioreactor-based assay to assess the effects of potential inhibitors on several enzymes simultaneously should prove useful for the survey of candidate inhibitors. To approach this goal, and as a proof of concept, we employed enzymes from the Gram-positive pathogen Enterococcus faecalis. Purified recombinant enzymes that catalyze the first three reactions of the mevalonate pathway were immobilized in two kinds of continuous flow enzyme bioreactors: a classical hollow fiber bioreactor and an immobilized plug flow bioreactor that exploited a novel method of enzyme immobilization. Both bioreactor types employed recombinant acetoacetyl-CoA thiolase, HMG-CoA synthase, and HMG-CoA reductase from E. faecalis to convert acetyl-CoA to mevalonate, the central intermediate of the mevalonate pathway. Reactor performance was monitored continuously by spectrophotometric measurement of the concentration of NADPH in the reactor effluent. Additional potential applications of an Ni(++) affinity support bioreactor include using recombinant enzymes from extremophiles for biosynthetic applications. Finally, linking a Ni(++) affinity support bioreactor to an HPLC-mass spectrometer would provide an experimental and pedagogical tool for study of metabolite flux and pool sizes of intermediates to model regulation in intact cells.     

Investigating the role of native propionyl‐CoA and methylmalonyl‐CoA metabolism on heterologous polyketide production in Escherichia coli

6‐Deoxyerythronolide B (6dEB) is the macrocyclic aglycone precursor of the antibiotic natural product erythromycin. Heterologous production of 6dEB in Escherichia coli was accomplished, in part, by designed over‐expression of a native prpE gene (encoding a propionyl‐CoA synthetase) and heterologous pcc genes (encoding a propionyl‐CoA carboxylase) to supply the needed propionyl‐CoA and (2S)‐methylmalonyl‐CoA biosynthetic substrates. Separate E. coli metabolism includes three enzymes, Sbm (a methylmalonyl‐CoA mutase), YgfG (a methylmalonyl‐CoA decarboxylase), and YgfH (a propionyl‐CoA:succinate CoA transferase), also involved in propionyl‐CoA and methylmalonyl‐CoA metabolism. In this study, the sbm, ygfG, and ygfH genes were individually deleted and over‐expressed to investigate their effect on heterologous 6dEB production. Our results indicate that the deletion and over‐expression of sbm did not influence 6dEB production; ygfG over‐expression reduced 6dEB production by fourfold while ygfH deletion increased 6dEB titers from 65 to 129 mg/L in shake flask experiments. It was also found that native E. coli metabolism could support 6dEB biosynthesis in the absence of exogenous propionate and the substrate provision pcc genes. Lastly, the effect of the ygfH deletion was tested in batch bioreactor cultures in which 6dEB titers improved from 206 to 527 mg/L. Biotechnol. Bioeng. 2010; 105: 567–573. © 2009 Wiley Periodicals, Inc.     

High resolution crystal structures of unliganded and liganded human liver ACBP reveal a new mode of binding for the acyl-CoA ligand

The acyl-CoA binding protein (ACBP) is essential for the fatty acid metabolism, membrane structure, membrane fusion, and ceramide synthesis. Here high resolution crystal structures of human cytosolic liver ACBP, unliganded and liganded with a physiological ligand, myristoyl-CoA are described. The binding of the acyl-CoA molecule induces only few structural differences near the binding pocket. The crystal form of the liganded ACBP, which has two ACBP molecules in the asymmetric unit, shows that in human ACBP the same acyl-CoA binding pocket is present as previously described for the bovine and Plasmodium falciparum ACBP and the mode of binding of the 3'-phosphate-AMP moiety is conserved. Unexpectedly, in one of the acyl-CoA binding pockets the acyl moiety is bound in a reversed mode as compared with the bovine and P. falciparum structures. In this binding mode, the myristoyl-CoA molecule is fully ordered and bound across the two ACBP molecules of the crystallographic asymmetric unit: the 3'-phosphate-AMP moiety is bound in the binding pocket of one ACBP molecule and the acyl chain is bound in the pocket of the other ACBP molecule. The remaining binding pocket cavities of these two ACBP molecules are filled by other ligand fragments. This novel binding mode shows that the acyl moiety can flip out of its classical binding pocket and bind elsewhere, suggesting a mechanism for the acyl-CoA transfer between ACBP and the active site of a target enzyme. This mechanism is of possible relevance for the in vivo function of ACBP.     

Hepatic enzymatic synthesis and hydrolysis of CoA esters of solvent-derived oxa acids

Many ethylene glycol-derived solvents are oxidized to xenobiotic alkoxyacetic acids (3-oxa acids) by hepatic enzymes. The toxicity of these ubiquitous solvents has been associated with their oxa acid metabolites. For many xenobiotic carboxylic acids, the toxicity is associated with the CoA ester of the acid. In this study, related alkoxyacetic acids were evaluated as potential substrates for acyl-CoA synthetases found in mitochondrial, peroxisomal, and microsomal fractions isolated from rat liver. Likewise, chemically synthesized oxa acyl-CoAs were used as substrates for acyl-CoA hydrolases associated with the same rat liver fractions. Activities of the xenobiotic oxygen-substituted substrates were compared with analogous physiologic aliphatic substrates by UV-vis spectrophotometric methods. All of the solvent-derived oxa acids were reasonable substrates for the acyl-CoA synthetases, although their activity was usually less than the corresponding physiologic acid. Acyl-CoA hydrolase activities were decreased compared with acyl-CoA synthetase activities for all substrates, especially for the oxa acyl-CoAs. These studies suggest that these xenobiotic carboxylic acids may be converted to reactive acyl-CoA moieties which will persist in areas of the cell proximal to lipid synthesis, beta-oxidation, protein acylation, and amino acid conjugation. The interaction of these xenobiotic acyl-CoAs with those processes may be important to their toxicity and/or detoxification.     

The active site and substrates binding mode of malonyl-CoA synthetase determined by transferred nuclear Overhauser effect spectroscopy, site-directed mutagenesis, and comparative modeling studies

The active sites and substrate bindings of Rhizobium trifolii molonyl-CoA synthetase (MCS) catalyzing the malonyl-CoA formation from malonate and CoA have been determined based on NMR spectroscopy, site-directed mutagenesis, and comparative modeling methods. The MCS-bound conformation of malonyl-CoA was determined from two-dimensional-transferred nuclear Overhauser effect spectroscopy data. MCS protein folds into two structural domains and consists of 16 alpha-helices, 24 beta-strands, and several long loops. The core active site was determined as a wide cleft close to the end of the small C-terminal domain. The catalytic substrate malonate is placed between ATP and His206 in the MCS enzyme, supporting His206 in its catalytic role as it generates reaction intermediate, malonyl-AMP. These findings are strongly supported by previous biochemical data, as well as by the site-directed mutagenesis data reported here. This structure reveals the biochemical role as well as the substrate specificity that conservative residues of adenylate-forming enzymes have.     

Characterization of triacsin C inhibition of short-, medium-, and long-chain fatty acid: CoA ligases of human liver

Short-, medium-, and long-chain fatty acid:CoA ligases from human liver were tested for their sensitivity to inhibition by triacsin C. The short-chain fatty acid:CoA ligase was inhibited less than 10% by concentrations of triacsin C as high as 80 microM. The two mitochondrial xenobiotic/medium-chain fatty acid:CoA ligases (XM-ligases), HXM-A and HXM-B, were partially inhibited by triacsin C, and the inhibitions were characterized by low affinity for triacsin C (K(I) values > 100 microM). These inhibitions were found to be the result of triacsin C competing with medium-chain fatty acid for binding at the active site. The microsomal and mitochondrial forms of long-chain fatty acid:CoA ligase (also termed long-chain fatty acyl-CoA synthetase, or long-chain acyl-CoA synthetase LACS) were potently inhibited by triacsin C, and the inhibition had identical characteristics for both LACS forms. Dixon plots of this inhibition were biphasic. There is a high-affinity site with a K(I) of 0.1 microM that accounts for a maximum of 70% of the inhibition. There is also a low affinity site with a K(I) of 6 microM that accounts for a maximum of 30% inhibition. Kinetic analysis revealed that the high-affinity inhibition of the mitochondrial and microsomal LACS forms is the result of triacsin C binding at the palmitate substrate site.The high-affinity triacsin C inhibition of both the mitochondrial and microsomal LACS forms was found to require a high concentration of free Mg(2+), with the EC(50) for inhibition being 3 mM free Mg(2+). The low affinity triacsin C inhibition was also enhanced by Mg(2+). The data suggests that Mg(2+) promotes triacsin C inhibition of LACS by enhancing binding at the palmitate binding site. In contrast, the partial inhibition of the XM-ligases by triacsin C, which showed only a low-affinity component, did not require Mg(2+).     

Aldehyde-alcohol dehydrogenase and/or thiolase overexpression coupled with CoA transferase downregulation lead to higher alcohol titers and selectivity in Clostridium acetobutylicum fermentations

Metabolic engineering (ME) of Clostridium acetobutylicum has led to increased solvent (butanol, acetone, and ethanol) production and solvent tolerance, thus demonstrating that further efforts have the potential to create strains of industrial importance. With recently developed ME tools, it is now possible to combine genetic modifications and thus implement more advanced ME strategies. We have previously shown that antisense RNA (asRNA)-based downregulation of CoA transferase (CoAT, the first enzyme in the acetone-formation pathway) results in increased butanol to acetone selectivity, but overall reduced butanol yields and titers. In this study the alcohol/aldehyde dehydrogenase (aad) gene (encoding the bifunctional protein AAD responsible for butanol and ethanol production from butyryl-CoA and acetyl-CoA, respectively) was expressed from the phosphotransbutyrylase (ptb) promoter to enhance butanol formation and selectivity, while CoAT downregulation was used to minimize acetone production. This led to early production of high alcohol (butanol plus ethanol) titers, overall solvent titers of 30 g/L, and a higher alcohol/acetone ratio. Metabolic flux analysis revealed the likely depletion of butyryl-CoA. In order to increase then the flux towards butyryl-CoA, we examined the impact of thiolase (THL, thl) overexpression. THL converts acetyl-CoA to acetoacetyl-CoA, the first step of the pathway from acetyl-CoA to butyryl-CoA, and thus, combining thl overexpression with aad overexpression decreased, as expected, acetate and ethanol production while increasing acetone and butyrate formation. thl overexpression in strains with asRNA CoAT downregulation did not significantly alter product formation thus suggesting that a more complex metabolic engineering strategy is necessary to enhance the intracellular butyryl-CoA pool and reduce the acetyl-CoA pool in order to achieve improved butanol titers and selectivity.     

Skeletal muscle fat oxidation is increased in African-American and white women after 10 days of endurance exercise training

Objective: Obesity is associated with lower rates of skeletal muscle fatty acid oxidation (FAO), which is linked to insulin resistance. FAO is reduced further in obese African‐American (AAW) vs. white women (CW) and may also be lower in lean AAW vs. CW. In lean CW, endurance exercise training (EET) elevates the oxidative capacity of skeletal muscle. Therefore, we determined whether EET would elevate skeletal muscle FAO similarly in AAW and CW with a lower lipid oxidative capacity. Research Methods and Procedures: In vitro rates of FAO were assessed in rectus abdominus muscle strips using [1‐¹⁴C] palmitate (Pal) from lean AAW [BMI = 24.2 ± 0.9 (standard error) kg/m²] and CW (23.6 ± 0.8 kg/m²) undergoing voluntary abdominal surgery. Lean AAW (22 ± 0.9 kg/m²) and CW (24 ± 0.8 kg/m²) and obese AAW (36 ± 1.2 kg/m²) and CW (40 ± 1.3 kg/m²) underwent 10 consecutive days of EET on a cycle ergometer (60 min/d, 75% peak oxygen uptake). FAO was measured in vastus lateralis homogenates as captured ¹⁴CO₂ using [1‐¹⁴C] Pal, palmitoyl‐CoA (Pal‐CoA), and palmityl‐carnitine (Pal‐Car). Results: Muscle strip experiments showed suppressed rates of FAO (p = 0.03) in lean AAW vs. CW. EET increased the rates of skeletal muscle Pal oxidation (p = 0.05) in both lean AAW and CW. In obese subjects, Pre‐EET Pal (but not Pal‐CoA or Pal‐Car) oxidation was lower (p = 0.05) in AAW vs. CW. EET increased Pal oxidation 100% in obese AAW (p < 0.05) and 59% (p < 0.05) in obese CW. Similar increases (p < 0.05) in post‐EET FAO were observed for Pal‐CoA and Pal‐Car in both groups. Discussion: Both lean and obese AAW possess a lower capacity for skeletal muscle FAO, but EET increases FAO similarly in both AAW and CW. These data suggest the use of EET for treatment against obesity and diabetes for both AAW and CW.     

Identification of the active site residues in ATP‐citrate lyase's carboxy‐terminal portion

ATP‐citrate lyase (ACLY) catalyzes production of acetyl‐CoA and oxaloacetate from CoA and citrate using ATP. In humans, this cytoplasmic enzyme connects energy metabolism from carbohydrates to the production of lipids. In certain bacteria, ACLY is used to fix carbon in the reductive tricarboxylic acid cycle. The carboxy(C)‐terminal portion of ACLY shows sequence similarity to citrate synthase of the tricarboxylic acid cycle. To investigate the roles of residues of ACLY equivalent to active site residues of citrate synthase, these residues in ACLY from Chlorobium limicola were mutated, and the proteins were investigated using kinetics assays and biophysical techniques. To obtain the crystal structure of the C‐terminal portion of ACLY, full‐length C. limicola ACLY was cleaved, first non‐specifically with chymotrypsin and subsequently with Tobacco Etch Virus protease. Crystals of the C‐terminal portion diffracted to high resolution, providing structures that show the positions of active site residues and how ACLY tetramerizes.     

Decreased Long-Chain Fatty Acyl CoA Elongation Activity in Quaking and Jimpy Mouse Brain: Deficiency in One Enzyme or Multiple Enzyme Activities?

Using long-chain fatty acyl CoAs (arachidoyl CoA and behenoyl CoA), a decrease in overall fatty acid chain elongation activity was observed in the quaking and jimpy mouse brain microsomes relative to controls. Arachidoyl CoA (20:0) and behenoyl CoA (22:0) elongation activities were depressed to about 50% and 80% of control values in quaking and jimpy mice, respectively. Measurement of the individual enzymatic activities of the elongation system revealed a single deficiency in enzyme activity; only the condensation activity was reduced to the same extent as total elongation in both quaking and jimpy mice. The activities of the other three enzymes, beta-ketoacyl CoA reductase, beta-hydroxyacyl CoA dehydrase, and trans-2-enoyl CoA reductase, in both mutants were similar to the activities present in the control mouse. In addition, the activities of these three enzymes were more than two to three orders of magnitude greater than the condensing enzyme activity in all three groups, establishing that the condensing enzyme catalyzes the rate-limiting reaction step of total elongation. When the elongation of palmitoyl CoA was measured, only a 25% decrease in total elongation occurred in both mutants; a similar percent decrease in the condensation of palmitoyl CoA also was observed. The activities of the other three enzymes were unaffected. These results support the concept of either multiple elongation pathways or multiple condensing enzymes.     

Arabidopsis ketoacyl‐CoA synthase 16 (KCS16) forms C36/C38 acyl precursors for leaf trichome and pavement surface wax

The aliphatic waxes sealing plant surfaces against environmental stress are generated by fatty acid elongase complexes, each containing a β‐ketoacyl‐CoA synthase (KCS) enzyme that catalyses a crucial condensation forming a new C─C bond to extend the carbon backbone. The relatively high abundance of C₃₅ and C₃₇ alkanes derived from C₃₆ and C₃₈ acyl‐CoAs in Arabidopsis leaf trichomes (relative to other epidermis cells) suggests differences in the elongation machineries of different epidermis cell types, possibly involving KCS16, a condensing enzyme expressed preferentially in trichomes. Here, KCS16 was found expressed primarily in Arabidopsis rosette leaves, flowers and siliques, and the corresponding protein was localized to the endoplasmic reticulum. The cuticular waxes on young leaves and isolated leaf trichomes of ksc16 loss‐of‐function mutants were depleted of C₃₅ and C₃₇ alkanes and alkenes, whereas expression of Arabidopsis KCS16 in yeast and ectopic overexpression in Arabidopsis resulted in accumulation of C₃₆ and C₃₈ fatty acid products. Taken together, our results show that KCS16 is the sole enzyme catalysing the elongation of C₃₄ to C₃₈ acyl‐CoAs in Arabidopsis leaf trichomes and that it contributes to the formation of extra‐long compounds in adjacent pavement cells.     

Improved polyhydroxybutyrate production by Saccharomyces cerevisiae through the use of the phosphoketolase pathway

The metabolic pathways of the central carbon metabolism in Saccharomyces cerevisiae are well studied and consequently S. cerevisiae has been widely evaluated as a cell factory for many industrial biological products. In this study, we investigated the effect of engineering the supply of precursor, acetyl‐CoA, and cofactor, NADPH, on the biosynthesis of the bacterial biopolymer polyhydroxybutyrate (PHB), in S. cerevisiae. Supply of acetyl‐CoA was engineered by over‐expression of genes from the ethanol degradation pathway or by heterologous expression of the phophoketolase pathway from Aspergillus nidulans. Both strategies improved the production of PHB. Integration of gapN encoding NADP⁺‐dependent glyceraldehyde‐3‐phosphate dehydrogenase from Streptococcus mutans into the genome enabled an increased supply of NADPH resulting in a decrease in glycerol production and increased production of PHB. The strategy that resulted in the highest PHB production after 100 h was with a strain harboring the phosphoketolase pathway to supply acetyl‐CoA without the need of increased NADPH production by gapN integration. The results from this study imply that during the exponential growth on glucose, the biosynthesis of PHB in S. cerevisiae is likely to be limited by the supply of NADPH whereas supply of acetyl‐CoA as precursor plays a more important role in the improvement of PHB production during growth on ethanol. Biotechnol. Bioeng. 2013; 110: 2216–2224. © 2013 Wiley Periodicals, Inc.     

Recognized sequence and conformation in design of linear peptides as a competitive inhibitor for HMG-CoA reductase

This study is an attempt to develop a simple search method for lead peptide candidates, which include constrained structures in a recognized sequence, using the design of a competitive inhibitor for HMG-CoA reductase (HMGR). A structure-functional analysis of previously synthesized peptides proposes that a competitive inhibitory peptide can be designed by maintaining bioactive conformation in a recognized sequence. A conformational aspect of the structure-based approach was applied to the peptide design. By analysis of the projections obtained through a principle component analysis (PCA) for short linear and cyclic peptides, a head-to-tail peptide cycle is considered as a model for its linear analogy. It is proposed that activities of the linear peptides based on an identical amino acid sequence, which are obtained from a less flexible peptide cycle, would be relatively higher than those obtained from more flexible cyclic peptides. The design criterion was formulated in terms of a 'V' parameter, reflecting a relative deviation of an individual peptide cycle from an average statistical peptide cycle based on all optimized structures of the cyclic peptides in set. Twelve peptide cycles were selected for the peptide library. Comparing the calculated 'V' parameters, two cyclic peptides (GLPTGG and GFPTGG) were selected as lead cycles from the library. Based on these sequences, six linear peptides obtained by breaking the cycle at different positions were selected as lead peptide candidates. The linear GFPTGG peptide, showing the highest inhibitory activity against HMGR, increases the inhibitory potency nearly tenfold. Kinetic analysis reveals that the GFPTGG peptide is a competitive inhibitor of HMG-CoA with an equilibrium constant of inhibitor binding (K(i)) of 6.4 +/- 0.3 microM. Conformational data support a conformation of the designed peptides close to the bioactive conformation of the previously synthesized active peptides.     

Acetyl coenzyme A synthetase catalyzed reactions of coenzyme A with α,β-unsaturated carboxylic acids

α,β-Unsaturated coenzyme A (CoA) thioesters including acrylyl CoA, methacrylyl CoA, and propiolyl CoA were synthesized by catalysis with acetyl CoA synthetase (EC 6.2.1.1). After isolation from the enzymatic reactions, the products were found to be the result of 1,4 addition of CoASH to the double bond and addition of water to the triple bond of the initial acyl CoA adducts. Structural determinations of these products by ¹H NMR, ¹³C NMR, and the chemical reactions leading to their formation are described.     

piRNA‐63076 contributes to pulmonary arterial smooth muscle cell proliferation through acyl‐CoA dehydrogenase

Piwi‐interacting RNAs (piRNAs) are thought to be germline‐specific and to be involved in maintaining genome stability during development. Recently, piRNA expression has been identified in somatic cells in diverse organisms. However, the roles of piRNAs in pulmonary arterial smooth muscle cell (PASMC) proliferation and the molecular mechanism underlying the hypoxia‐regulated pathological process of pulmonary hypertension are not well understood. Using hypoxic animal models, cell and molecular biology, we obtained the first evidence that the expression of piRNA‐63076 was up‐regulated in hypoxia and was positively correlated with cell proliferation. Subsequently, we showed that acyl‐CoA dehydrogenase (Acadm), which is negatively regulated by piRNA‐63076 and interacts with Piwi proteins, was involved in hypoxic PASMC proliferation. Finally, Acadm inhibition under hypoxia was partly attributed to DNA methylation of the Acadm promoter region mediated by piRNA‐63076. Overall, these findings represent invaluable resources for better understanding the role of epigenetics in pulmonary hypertension associated with piRNAs.