Malate synthase activity in cotton and other ungerminated oilseeds: a survey

Extracts from several species and varieties of ungerminated cotton seeds plus homogenates from 18 other oilseeds (representing 11 different families) were examined for malate synthase and isocitrate lyase activity. Malate synthase activities in the various cotton seeds ranged from 35 to 129% of the units per dry seed weight found in Deltapine 16 cotton. For other oilseeds, the range was from 0.3 to 58% of Deltapine 16 cotton. Castor bean (Ricinus communis L.) had the least activity per mg dry weight (12-fold lower than the next lowest species), while Pima cotton (Gossypium barbadense L.) had the highest level (8.53 units). On a per seed basis, these values were 15 and 747 nanomoles per minute.     

Mechanism of the intramolecular Claisen condensation reaction catalyzed by MenB, a crotonase superfamily member

MenB, the 1,4-dihydroxy-2-naphthoyl-CoA synthase from the bacterial menaquinone biosynthesis pathway, catalyzes an intramolecular Claisen condensation (Dieckmann reaction) in which the electrophile is an unactivated carboxylic acid. Mechanistic studies on this crotonase family member have been hindered by partial active site disorder in existing MenB X-ray structures. In the current work the 2.0 ? structure of O-succinylbenzoyl-aminoCoA (OSB-NCoA) bound to the MenB from Escherichia coli provides important insight into the catalytic mechanism by revealing the position of all active site residues. This has been accomplished by the use of a stable analogue of the O-succinylbenzoyl-CoA (OSB-CoA) substrate in which the CoA thiol has been replaced by an amine. The resulting OSB-NCoA is stable, and the X-ray structure of this molecule bound to MenB reveals the structure of the enzyme-substrate complex poised for carbon-carbon bond formation. The structural data support a mechanism in which two conserved active site Tyr residues, Y97 and Y258, participate directly in the intramolecular transfer of the substrate α-proton to the benzylic carboxylate of the substrate, leading to protonation of the electrophile and formation of the required carbanion. Y97 and Y258 are also ideally positioned to function as the second oxyanion hole required for stabilization of the tetrahedral intermediate formed during carbon-carbon bond formation. In contrast, D163, which is structurally homologous to the acid-base catalyst E144 in crotonase (enoyl-CoA hydratase), is not directly involved in carbanion formation and may instead play a structural role by stabilizing the loop that carries Y97. When similar studies were performed on the MenB from Mycobacterium tuberculosis, a twisted hexamer was unexpectedly observed, demonstrating the flexibility of the interfacial loops that are involved in the generation of the novel tertiary and quaternary structures found in the crotonase superfamily. This work reinforces the utility of using a stable substrate analogue as a mechanistic probe in which only one atom has been altered leading to a decrease in α-proton acidity.     

Whole-brain glutamate metabolism evaluated by steady-state kinetics using a double-isotope procedure: effects of gabapentin

Cerebral rates of anaplerosis are known to be significant, yet the rates measured in vivo have been debated. In order to track glutamate metabolism in brain glutamatergic neurons and brain glia, for the first time unrestrained awake rats were continuously infused with a combination of H14CO3- and [1 - 13C]glucose in over 50 infusions ranging from 5 to 60 min. In whole-brain extracts from these animals, the appearance of 14C in brain glutamate and glutamine and appearance of 13C in the C-4 position of glutamate and glutamine were measured as a function of time. The rate of total neuronal glutamate turnover, the anaplerotic rate of synthesis of glutamine and glutamate from H14CO3-, flux through the glutamate/glutamine cycle, and a minimum estimate of whole-brain anaplerosis was obtained. The rate of synthesis of 14C-glutamate from H14CO3- was 1.29 +/- 0.11 nmoles/min/mg protein, whereas the rate of synthesis of 14C-glutamine was 1.48 +/- 0.10 nmoles/min/mg protein compared to total glutamate turnover of 9.39 +/- 0.73 nmoles/min/mg protein. From the turnover rate of glutamine, an upper limit for flux through the glutamate/glutamine cycle was estimated at 4.6 nmoles/min/mg protein. Synthesis of glutamine from H14CO3- was substantial, amounting to 32% of the glutamate/glutamine cycle. These rates were not significantly affected by a single injection of 100 mg/kg of the antiepileptic drug gabapentin. In contrast, acute administration of gabapentin significantly lowered incorporation of H14CO3- into glutamate and glutamine in excised rat retinas, suggesting metabolic effects of gabapentin may require chronic treatment and/or are restricted to brain areas enriched in target enzymes such as the cytosolic branched chain aminotransferase. We conclude that the brain has a high anaplerotic activity and that the combination of two tracers with different precursors affords unique insights into the compartmentation of cerebral metabolism.     

Purification and characterization of OleA from Xanthomonas campestris and demonstration of a non-decarboxylative Claisen condensation reaction

OleA catalyzes the condensation of fatty acyl groups in the first step of bacterial long-chain olefin biosynthesis, but the mechanism of the condensation reaction is controversial. In this study, OleA from Xanthomonas campestris was expressed in Escherichia coli and purified to homogeneity. The purified protein was shown to be active with fatty acyl-CoA substrates that ranged from C(8) to C(16) in length. With limiting myristoyl-CoA (C(14)), 1 mol of the free coenzyme A was released/mol of myristoyl-CoA consumed. Using [(14)C]myristoyl-CoA, the other products were identified as myristic acid, 2-myristoylmyristic acid, and 14-heptacosanone. 2-Myristoylmyristic acid was indicated to be the physiologically relevant product of OleA in several ways. First, 2-myristoylmyristic acid was the major condensed product in short incubations, but over time, it decreased with the concomitant increase of 14-heptacosanone. Second, synthetic 2-myristoylmyristic acid showed similar decarboxylation kinetics in the absence of OleA. Third, 2-myristoylmyristic acid was shown to be reactive with purified OleC and OleD to generate the olefin 14-heptacosene, a product seen in previous in vivo studies. The decarboxylation product, 14-heptacosanone, did not react with OleC and OleD to produce any demonstrable product. Substantial hydrolysis of fatty acyl-CoA substrates to the corresponding fatty acids was observed, but it is currently unclear if this occurs in vivo. In total, these data are consistent with OleA catalyzing a non-decarboxylative Claisen condensation reaction in the first step of the olefin biosynthetic pathway previously found to be present in at least 70 different bacterial strains.     

Enzymatic condensation of dopamine and acetaldehyde: a salsolinol synthase from rat brain

Salsolinol (1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline; Sal) is structurally similar to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, which is supposed to have a role in the development of Parkinson-like syndrome in both human and non-human subjects. In the human brain, the amount of (R)-enantiomer of Sal is much higher than (S)-enantiomer, suggesting that a putative enzyme may participate in the synthesis of (R)-salsolinol, called (R)-salsolinol synthase. In this study, the (R)-salsolinol synthase activity in the condensation of dopamine and acetaldehyde was investigated in the crude extracts from the brains of Sprague Dawley rats. Identification of the enzymatic reaction products and enzyme activity detection were achieved by HPLC-electrochemical detection. The discovery of this enzyme activity in rat’s brain indicates the natural existence of (R)-salsolinol synthase in the brains of humans and rats, and it is distributed in most brain regions of rat with higher activity in soluble proteins extracted from striatum and substantia nigra.     

Enzymatic proof for the identity of the S-sulfocysteine synthase and cysteine synthase B of Salmonella typhimurium.

S-Sulfocysteine synthase was isolated from Salmonella typhimurium LT-2 to homogeneous form with polyacrylamide gel electrophoresis. The molecular weight of this enzyme was determined to be ca. 55,000. The enzyme consisted of two identically sized subunits, and it contained one pyridoxal phosphate per subunit. The enzyme catalyzed the biosynthesis of cysteine or S-methylcysteine from sulfide or methanethiol and O-acetylserine, respectively, in addition to the formation of S-sulfocysteine from thiosulfate and O-acetylserine. The enzyme is identical to cysteine synthase B. The intracellular level of this enzyme was regulated by lesser extents of the same factors as those effective for cysteine synthase A.     

Role of a conserved arginine in the mechanism of acetohydroxyacid synthase: catalysis of condensation with a specific ketoacid substrate

The thiamin diphosphate (ThDP)-dependent bio-synthetic enzyme acetohydroxyacid synthase (AHAS) catalyzes decarboxylation of pyruvate and specific condensation of the resulting ThDP-bound two-carbon intermediate, hydroxyethyl-ThDP anion/enamine (HEThDP(-)), with a second ketoacid, to form acetolactate or acetohydroxybutyrate. Whereas the mechanism of formation of HEThDP(-) from pyruvate is well understood, the role of the enzyme in control of the carboligation reaction of HEThDP(-) is not. Recent crystal structures of yeast AHAS from Duggleby's laboratory suggested that an arginine residue might interact with the second ketoacid substrate. Mutagenesis of this completely conserved residue in Escherichia coli AHAS isozyme II (Arg(276)) confirms that it is required for rapid and specific reaction of the second ketoacid. In the mutant proteins, the normally rapid second phase of the reaction becomes rate-determining. A competing alternative nonnatural but stereospecific reaction of bound HEThDP(-) with benzaldehyde to form phenylacetylcarbinol (Engel, S., Vyazmensky, M., Geresh, S., Barak, Z., and Chipman, D. M. (2003) Biotechnol. Bioeng. 84, 833-840) provides a new tool for studying the fate of HEThDP(-) in AHAS, since the formation of the new product has a very different dependence on active site modifications than does acetohydroxyacid acid formation. The effects of mutagenesis of four different residues in the site on the rates and specificities of the normal and unnatural reactions support a critical role for Arg(276) in the stabilization of the transition states for ligation of the incoming second ketoacid with HEThDP(-) and/or for the breaking of the product-ThDP bond. This information makes it possible to engineer the active site so that it efficiently and preferentially catalyzes a new reaction.     

Malate dehydrogenase: a useful phylogenetic marker for the genus Aeromonas

The reconstruction of correct genealogies among biological entities, the estimation of the divergence time between organisms or the study of the different events that occur along evolutionary lineages are not always based on suitable genes. For reliable results, it is necessary to look at full-length sequences of genes under stabilizing selection (neutral or purifying) and behaving as good molecular clocks. In bacteria it has been proved that the malate dehydrogenase gene (mdh) can be used to determine the inter- and intraspecies divergence, and hence this gene constitutes a potential marker for phylogeny and bacterial population genetics. We have sequenced the full-length mdh gene in 36 type and reference strains of Aeromonas. The species grouping obtained in the phylogenetic tree derived from mdh sequences was in agreement with that currently accepted for the genus Aeromonas. The maximum likelihood models applied to our sequences indicated that the mdh gene is highly conserved among the Aeromonas species and the main evolutionary force acting on it is purifying selection. Only two sites under potential diversifying selection were identified (T 108 and S 193). In order to determine if these two residues could have an influence on the MDH structure, we mapped them in a three-dimensional model constructed from the sequence of A. hydrophila using the human mitochondrial MDH as a template. The presence of purifying selection together with the linear relationship between substitutions and gene divergence makes the mdh an excellent candidate gene for a phylogeny of Aeromonas and probably for other bacterial groups.     

Malate Utilization by a Group D Streptococcus: Regulation of Malic Enzyme Synthesis by an Inducible Malate Permease

Induction of an nicotinamide adenine dinucleotide-specific malic enzyme and a malate entry system permits Streptococcus faecalis to grow at the expense of malate. Evidence is presented which shows that biosynthesis of the permease, but not of the malic enzyme, is subject to catabolite repression by glucose. In contrast to the malic enzyme, the catalytic function of the entry system does not appear to be inhibited by intermediate products of glycolysis. Although the induction of the entry system does not appear to be coordinated with the induction of the malic enzyme, the latter process is dependent upon the permease for the transport and accumulation of inducer.     

Quantitative investigations of cell-bubble interactions using a foam fractionation technique

Previous work by the authors and others has shown that suspended animal cell damage in bioreactors is caused by cell-bubble interactions, regardless whether the bubbles are from bubble entrainment or direct gas sparging. As approach to measure the adsorptivity of animal cells to bubbles, a modified batch foam fractionation technique has been developed in this work and proven to be applicable. By using this technique, the number of cells adsorbed per unit bubble surface area and the adsorption coefficients have been measured to quantify hybridoma cell-bubble interactions, and the prevetive effects of serum and Pluronic F68 on these interactions. It was demonstrated quantitatively that the hybridoma cells adhere to bubbles spontaneously and significant numbers exist in the foam, and that both the serum and Pluronic F68 provide strong prevention to these cell-bubble interactions. The results obtained provide criteria for bioreactor operation and medium formulation to prevent cell-bubble interactions and cell damage in the culture processes.Abbreviations NBCS new born calf serum - SFM serum-free medium     

Isolation of viral double-stranded RNAs using a LiCl fractionation procedure

A general procedure for the isolation of virus-specific double-stranded RNA (ds-RNA) is discribed. The procedure is based on the differential solubility of different types of nucleic acids in LiCl. Principal advantages over conventional methods are simplicity, avoidance of enzymatic treatment, and relatively good yields of undegraded ds-RNA while permitting separation of several main groups of cellular and viral nucleic acids from the same batch of tissue. The method has been successfully applied in tissues infected by several representative plant RNA viruses. The virus-specific ds-RNAs obtained have been identified by their resistance to ribonuclease and comparison of their electrophoretic mobilities with those of the corresponding single-stranded RNA (ss-RNA) in polyacrylamide gels. The molecular weights of the ds-RNAs of tobacco mosaic virus, turnip yellow mosaic virus, alfalfa mosaic virus, and peanut stunt virus fit the curved log molecular weight-migration relationship constructed from a set of known marker ds-RNAs.     

Energetics of proline racemase: double fractionation experiment, a test for concertedness and for transition-state dominance

To test whether a reaction involving the making and/or breaking of two bonds at two sites is concerted (and proceeds through a single transition state) or is stepwise (and involves a reaction intermediate in which only one bond has been made or broken), we have measured the isotopic fractionation at one site as a function of isotopic substitution at the other site. In the case of proline racemase, the discrimination against solvent deuterium in the product when the reaction is run in mixed H2O-D2O is measured for the reaction both of [2-1H]proline and of [2-2H]proline. The isotopic fractionation at the solvent site may in principle be smaller, the same, or larger, when the 2H-labeled substrate is used rather than the 1H substrate, and--depending upon the nature of the catalyzing groups--this information indicates whether the reaction is stepwise, or concerted, or whether an isotopically insensitive transition state is partially rate determining. Experimentally, we have found that the discrimination against solvent deuterium in the product L-proline is the same, whether D-[2-1H]proline or D-[2-2H]proline is the substrate. This result requires that the substrate and product "on-off" steps are faster than the racemization step and that the racemization reaction proceeds either in a concerted manner or in a stepwise fashion involving enzyme catalytic groups (e.g., thiols) having ground-state fractionation factors around 0.5.     

Ants find the shortest path: a mathematical proof

In the most basic application of Ant Colony Optimization (ACO), a set of artificial ants find the shortest path between a source and a destination. Ants deposit pheromone on paths they take, preferring paths that have more pheromone on them. Since shorter paths are traversed faster, more pheromone accumulates on them in a given time, attracting more ants and leading to reinforcement of the pheromone trail on shorter paths. This is a positive feedback process that can also cause trails to persist on longer paths, even when a shorter path becomes available. To counteract this persistence on a longer path, ACO algorithms employ remedial measures, such as using negative feedback in the form of uniform evaporation on all paths. Obtaining high performance in ACO algorithms typically requires fine tuning several parameters that govern pheromone deposition and removal. This paper proposes a new ACO algorithm, called EigenAnt, for finding the shortest path between a source and a destination, based on selective pheromone removal that occurs only on the path that is actually chosen for each trip. We prove that the shortest path is the only stable equilibrium for EigenAnt, which means that it is maintained for arbitrary initial pheromone concentrations on paths, and even when path lengths change with time. The EigenAnt algorithm uses only two parameters and does not require them to be finely tuned. Simulations that illustrate these properties are provided.     

Hysteretic behaviour of citrate synthase. Isotopically chiral citryl-CoA reveals increased number of reversals of the synthase condensation step under steady-state conditions

Citrate specimens derived from chiral acetates were converted to the CoA derivatives. These were reconverted with citrate synthase to citrates under conditions of either predominating hydrolytic burst or predominating steady-state period. The stereochemical purity of substrates and products was determined. Reversal of the synthase condensation step occurs under both conditions but is markedly increased during the steady-state period. The results indicate that citryl-CoA-derived acetyl-CoA and oxaloacetate create the steady-state conditions. The hydrolase state and the ligase/lyase state of the synthase predominate under burst and steady-state conditions, respectively. This result indicates a conversion of the hydrolase state into the ligase/lyase state during the transition.     

Modification of a neuronal network direction using stepwise photo-thermal etching of an agarose architecture

Control over spatial distribution of individual neurons and the pattern of neural network provides an important tool for studying information processing pathways during neural network formation. Moreover, the knowledge of the direction of synaptic connections between cells in each neural network can provide detailed information on the relationship between the forward and feedback signaling. We have developed a method for topographical control of the direction of synaptic connections within a living neuronal network using a new type of individual-cell-based on-chip cell-cultivation system with an agarose microchamber array (AMCA). The advantages of this system include the possibility to control positions and number of cultured cells as well as flexible control of the direction of elongation of axons through stepwise melting of narrow grooves. Such micrometer-order microchannels are obtained by photo-thermal etching of agarose where a portion of the gel is melted with a 1064-nm infrared laser beam. Using this system, we created neural network from individual Rat hippocampal cells. We were able to control elongation of individual axons during cultivation (from cells contained within the AMCA) by non-destructive stepwise photo-thermal etching. We have demonstrated the potential of our on-chip AMCA cell cultivation system for the controlled development of individual cell-based neural networks.     

Synthesis of monoacylglycerol containing pinolenic acid via stepwise esterification using a cold active lipase

High purity monoacylglycerol (MAG) containing pinolenic acid was synthesized via stepwise esterification of glycerol and fatty acids from pine nut oil using a cold active lipase from Penicillium camembertii as a biocatalyst. Effects of temperature, molar ratio, water content, enzyme loading, and vacuum on the synthesis of MAG by lipase‐catalyzed esterification of glycerol and fatty acid from pine nut oil were investigated. Diacylglycerol (DAG) as well as MAG increased significantly when temperature was increased from 20 to 40°C. At a molar ratio of 1:1, MAG content decreased because of the significant increase in DAG content. Water has a profound influence on both MAG and DAG content through the entire course of reaction. The reaction rate increased significantly as enzyme loading increased up to 600 units. Vacuum was an effective method to reduce DAG content. The optimum temperature, molar ratio, water content, enzyme loading, vacuum, and reaction time were 20°C, 1:5 (fatty acid to glycerol), 2%, 600 units, 5 torr, and 24 h, respectively. MAG content further increased via lipase‐catalyzed second step esterification at subzero temperature. P. camembertii lipase exhibited esterification activity up to ?30°C. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Optical fingerprinting of peptides using two-dimensional infrared spectroscopy: proof of principle

We employ a particular form of two-dimensional infrared four-wave mixing (2DIR FWM) as a vibrational spectroscopic tool to quantify the amino acid content of a number of peptides. Vibrational features corresponding to ring modes of the aromatic groups of phenylalanine (Phe) and tyrosine (Tyr), as well as a methylene mode that is used as an internal reference, are identified. We show that the ratios of the integrated intensities, and the amplitudes, of the aromatic peaks of Phe and Tyr relative to the methylene integrated intensity, and amplitude, are proportional to the actual ratio of Phe and Tyr to CH₂ in the samples within a precision of ±12.5%. This precision is shown to be sufficient to use this form of 2DIR spectroscopy as a possible proteins fingerprinting tool.     

New Attempt at the Synthesis of Lavandulol by a Claisen Type Rearrangement

The use of the Claisen type rearrangement previously studied by K. C. Brannock¹⁾ on some alkyl allyl esters leads in the present study from I, I-dimethyl-2-propenyl senecioate to a mixture of lavandulic acid 70% and iso-lavandulic acid 30%,