Individual Enzymatic Reactions Research Articles

Cell-Free Systems Biology: Characterizing Central Metabolism of Clostridium thermocellum with a Three-Enzyme Cascade Reaction.

Genetic approaches have been traditionally used to understand microbial metabolism, but this process can be slow in nonmodel organisms due to limited genetic tools. An alternative approach is to study metabolism directly in the cell lysate. This avoids the need for genetic tools and is routinely used to study individual enzymatic reactions but is not generally used to study systems-level properties of metabolism. Here we demonstrate a new approach that we call "cell-free systems biology", where we use well-characterized enzymes and multienzyme cascades to serve as sources or sinks of intermediate metabolites. This allows us to isolate subnetworks within metabolism and study their systems-level properties. To demonstrate this, we worked with a three-enzyme cascade reaction that converts pyruvate to 2,3-butanediol. Although it has been previously used in cell-free systems, its pH dependence was not well characterized, limiting its utility as a sink for pyruvate. We showed that improved proton accounting allowed better prediction of pH changes and that active pH control allowed 2,3-butanediol titers of up to 2.1 M (189 g/L) from acetoin and 1.6 M (144 g/L) from pyruvate. The improved proton accounting provided a crucial insight that preventing the escape of CO2 from the system largely eliminated the need for active pH control, dramatically simplifying our experimental setup. We then used this cascade reaction to understand limits to product formation in Clostridium thermocellum, an organism with potential applications for cellulosic biofuel production. We showed that the fate of pyruvate is largely controlled by electron availability and that reactions upstream of pyruvate limit overall product formation.

One‐Pot Production of Vanillin from Capsaicinoids through a Retrosynthetic Enzyme Cascade

AbstractVanillin is a key organoleptic component in vanilla, one of the most important natural flavors in the industry. The limited supply of natural vanilla, extracted from cured vanilla beans, elevates the commercial value of ‘natural’‐labeled vanillin, producible by microbial conversion of natural precursors such as ferulic acid, eugenol, isoeugenol, lignin, and glucose. However, cellular toxicity and undesirable overoxidation of vanillin remain challenges to the microbial methods. Here, we developed a one‐pot enzymatic conversion of capsaicinoids to vanillin by retracing the biosynthetic pathway found in Capsicum plants. The enzyme cascade consists of the hydrolysis of capsaicinoids by immobilized lipase B from Candida antarctica (CALB) and the oxidative deamination of the resulting vanillylamine by ω‐transaminase from Paracoccus denitrificans (PDTA). Process optimization of individual enzyme reactions was conducted on an analytical scale, enabling>95% reaction yield of vanillin from capsaicinoids in the one‐pot cascade reaction without undesirable accumulation of the oxidation byproduct and the inhibitory reaction intermediate, i. e., vanillic acid and vanillylamine, respectively. To verify practical applicability, we performed a preparative‐scale reaction starting with 1.75 g capsaicinoids and 2.75 g sodium pyruvate at 10 mg/mL CALB and 3 μM PDTA, leading to an 81% reaction yield and a 57% isolation yield. This study is, to the best of our knowledge, the first demonstration of one‐pot production of vanillin from capsaicinoids, which might pave the way for cost‐effective and sustainable access to natural‐labeled vanillin from a renewable feedstock readily available from Capsicum fruits.

Pushing the boundaries of phosphorylase cascade reaction for cellobiose production I: Kinetic model development.

One-pot cascade reactions of coupled disaccharide phosphorylases enable an efficient transglycosylation via intermediary α-d-glucose 1-phosphate (G1P). Such transformations have promising applications in the production of carbohydrate commodities, including the disaccharide cellobiose for food and feed use. Several studies have shown sucrose and cellobiose phosphorylase for cellobiose synthesis from sucrose, but the boundaries on transformation efficiency that result from kinetic and thermodynamic characteristics of the individual enzyme reactions are not known. Here, we assessed in a step-by-step systematic fashion the practical requirements of a kinetic model to describe cellobiose production at industrially relevant substrate concentrations of up to 600 mM sucrose and glucose each. Mechanistic initial-rate models of the two-substrate reactions of sucrose phosphorylase (sucrose + phosphate → G1P + fructose) and cellobiose phosphorylase (G1P + glucose → cellobiose + phosphate) were needed and additionally required expansion by terms of glucose inhibition, in particular a distinctive two-site glucose substrate inhibition of the cellobiose phosphorylase (from Cellulumonas uda). Combined with mass action terms accounting for the approach to equilibrium, the kinetic model gave an excellent fit and a robust prediction of the full reaction time courses for a wide range of enzyme activities as well as substrate concentrations, including the variable substoichiometric concentration of phosphate. The model thus provides the essential engineering tool to disentangle the highly interrelated factors of conversion efficiency in the coupled enzyme reaction; and it establishes the necessary basis of window of operation calculations for targeted optimizations toward different process tasks.

Guvermectin Biosynthesis Revealing the Key Role of a Phosphoribohydrolase and Structural Insight into the Active Glutamate of a Noncanonical Adenine Phosphoribosyltransferase.

Guvermectin is a novel plant growth regulator that has been registered as a new agrochemical in China. It is an adenosine analogue with an unusual psicofuranose instead of ribose. Herein, the gene cluster responsible for guvermectin biosynthesis in Streptomyces caniferus NEAU6 is identified using gene interruption and heterologous expression experiments. A key intermediate psicofuranine 6'-phosphate (PMP) is chemically synthesized, and the functions of GvmB, C, D, and E are verified by individual stepwise enzyme reactions in vitro. The results also show that the biosynthesis of guvermectin is coupled with adenosine production by a single cluster. The higher catalytic efficiency of GvmB on PMP than AMP ensures the effective biosynthesis of guvermectin. Moreover, a phosphoribohydrolase GvmA is employed in the pathway that can hydrolyze AMP but not PMP and shows higher catalytic efficiency for the AMP hydrolysis than that of the AMP dephosphorylation by GvmB, leading to shunting of adenosine biosynthesis toward the production of guvermectin. Finally, the crystal structure of GvmE in complex with the product PMP has been solved. Glu160 at the C-terminal is identified as the acid/base for protonation/deprotonation of N7 of the adenine ring, demonstrating that GvmE is a noncanonical adenine phosphoribosyltransferase.

Kinetics of Intracellular Electron Generation in Shewanella oneidensis MR-1.

Efficient utilization of bacterial bioresources requires quantitative evaluation of metabolic activity in living bacterial cells. Shewanella oneidensis MR-1 transfers electrons generated within the cell to the extracellular environment via the cytochrome complex in the inner/outer membranes and is one of the most useful bacteria for the recovery of metals, treatment of wastewater, and preparation of microbial fuel cells. Here, we performed a quantitative evaluation of electron generation based on individual enzyme reactions in S. oneidensis MR-1. By using potentiometric measurements, we have examined intracellular electron generation in bacterial suspensions of S. oneidensis supplemented with different carbon sources (formate, lactate, pyruvate, or acetyl coenzyme A) or ferricyanide, which was almost completely reduced to ferrocyanide during the incubation without affecting bacterial cell viability. The amount of electron generation strongly depended on the nature of the carbon source. Analysis of the obtained kinetic parameters of intracellular electron generation demonstrated that formate was the most effective carbon source, as it enabled 2.5-fold faster electron generation rate than other sources. We established that the respective contributions of lactate dehydrogenase, pyruvate dehydrogenase/pyruvate-formate-lyase, and tricarboxylic acid cycle to lactate metabolism were 62%, 31%, and 7.4%, correspondingly. Furthermore, we clarified that electrons may be generated at 1.6 × 10-12 A s-1 by ideal metabolism in a single living cell. These findings establish the basis for biological strategies of electron production and facilitate the utilization of S. oneidensis as a bioresource in practical applications, including energy production, environmental purification, and recovery of useful materials.

Explicit consideration of topological and parameter uncertainty gives new insights into a well‐established model of glycolysis

Previous models of glycolysis in the sleeping sickness parasite Trypanosoma brucei assumed that the core part of glycolysis in this unicellular parasite is tightly compartimentalized within an organelle, the glycosome, which had previously been shown to contain most of the glycolytic enzymes. The glycosomes were assumed to be largely impermeable, and exchange of metabolites between the cytosol and the glycosome was assumed to be regulated by specific transporters in the glycosomal membrane. This tight compartmentalization was considered to be essential for parasite viability. Recently, size-specific metabolite pores were discovered in the membrane of glycosomes. These channels are proposed to allow smaller metabolites to diffuse across the membrane but not larger ones. In light of this new finding, we re-analyzed the model taking into account uncertainty about the topology of the metabolic system in T. brucei, as well as uncertainty about the values of all parameters of individual enzymatic reactions. Our analysis shows that these newly-discovered nonspecific pores are not necessarily incompatible with our current knowledge of the glycosomal metabolic system, provided that the known cytosolic activities of the glycosomal enzymes play an important role in the regulation of glycolytic fluxes and the concentration of metabolic intermediates of the pathway.

Bio-recognition and functional lipidomics by glycosphingolipid transfer technology.

Through glycosphingolipid biochemical research, we developed two types of transcription technologies. One is a biochemical transfer of glycosphingolipids to peptides. The other is a physicochemical transfer of glycosphingolipids in silica gel to the surface of a plastic membrane. Using the first technology, we could prepare peptides which mimic the shapes of glycosphingolipid molecules by biopanning with a phage-displayed peptide library and anti-glycosphingolipid antibodies as templates. The peptides thus obtained showed biological properties and functions similar to those of the original glycosphingolipids, such as lectin binding, glycosidase modulation, inhibition of tumor metastasis and immune response against the original antigen glycosphingolipid, and we named them glyco-replica peptides. The results showed that the newly prepared peptides could be used effectively as a bio-recognition system and suggest that the glyco-replica peptides can be widely applied to therapeutic fields. Using the second technology, we could establish a functional lipidomics with a thin-layer chromatography-blot/matrix-assisted laser desorption ionization-time of flight mass spectrometry (TLC-Blot/MALDI-TOF MS) system. By transferring glycosphingolipids on a plastic membrane surface from a TLC plate, innovative biochemical approaches such as simple purification of individual glycosphingolipids, binding studies, and enzyme reactions could be developed. The combinations of these biochemical approaches and MALDI-TOF MS on the plastic membrane could provide new strategies for glycosphingolipid science and the field of lipidomics. In this review, typical applications of these two transfer technologies are introduced.

Sex steroids in Sjögren’s syndrome

The purpose of the review is to consider pathomechanisms of Sjögren’s syndrome (SS), which could explain the female dominance (9:1), the most common age of onset (40–50 years) and targeting of the exocrine glands. Estrogens seem to specifically protect secretory glandular acinar cells against apoptosis whereas lack of estrogens during menopause and climacterium specifically leads to increased apoptosis of the exocrine secretory cells. Male gonads produce testosterone and convert it in exocrine glands to dihydrotesterosterone (DHT), which is anti-apoptotic and protects against acinar cell apoptosis. Estrogen-deficient women need to produce dehydroepiandrosterone (DHEA) in the adrenal glands and convert it to DHT in exocrine glands in a complex and branching reaction network in which individual enzymatic reactions are catalyzed in forward and backward directions by a myriad of different isoforms of steroidogenic enzymes. Tailoring DHT in peripheral tissues is much more complex and vulnerable in women than in men. In SS the intracrine steroidogenic enzyme machinery is deranged. These endo-/intracrine changes impair acinar remodeling due to impaired integrin α1β1 and integrin α2β1 expression so that the intercalated duct progenitor cells are unable to migrate to the acinar space, to differentiate to secretory acinar cells upon contact with laminin-111 and laminin-211 specifically found in the acinar basement membrane. The disarranged endo-/intracrine estrogen/androgen balance induces acinar cells to release microparticles and apoptotic bodies and to undergo apoptotis and/or anoikis. Membrane particles contain potential autoantigens recognized by T- (TCRs) and B-cell receptors (BCRs) and danger-associated molecular patterns (DAMPs) recognized by Toll-like receptors (TLRs). In membrane particles (or carrier-complexes) antigen/adjuvant complexes could stimulate professional antigen capturing, processing and presenting cells, which can initiate auto-inflammatory and autoimmune cascades, break the self-tolerance and finally lead to SS.

Carbon and sulfur back flux during anaerobic microbial oxidation of methane and coupled sulfate reduction.

Microbial degradation of substrates to terminal products is commonly understood as a unidirectional process. In individual enzymatic reactions, however, reversibility (reverse reaction and product back flux) is common. Hence, it is possible that entire pathways of microbial degradation are associated with back flux from the accumulating product pool through intracellular intermediates into the substrate pool. We investigated carbon and sulfur back flux during the anaerobic oxidation of methane (AOM) with sulfate, one of the least exergonic microbial catabolic processes known. The involved enzymes must operate not far from the thermodynamic equilibrium. Such an energetic situation is likely to favor product back flux. Indeed, cultures of highly enriched archaeal-bacterial consortia, performing net AOM with unlabeled methane and sulfate, converted label from (14)C-bicarbonate and (35)S-sulfide to (14)C-methane and (35)S-sulfate, respectively. Back fluxes reached 5% and 13%, respectively, of the net AOM rate. The existence of catabolic back fluxes in the reverse direction of net reactions has implications for biogeochemical isotope studies. In environments where biochemical processes are close to thermodynamic equilibrium, measured fluxes of labeled substrates to products are not equal to microbial net rates. Detection of a reaction in situ by labeling may not even indicate a net reaction occurring in the direction of label conversion but may reflect the reverse component of a so far unrecognized net reaction. Furthermore, the natural isotopic composition of the substrate and product pool will be determined by both the forward and back flux. This finding may have to be considered in the interpretation of stable isotope records.

Microbially mediated re-oxidation of sulfide during dissimilatory sulfate reduction by Desulfobacter latus

Enzymatic reactions during dissimilatory sulfate reduction (DSR) are often treated as unidirectional with respect to dissolved sulfide. However, quantitative models describing kinetic sulfur isotope fractionations during DSR consider the individual enzymatic reactions as reversible ( Rees, 1973). Brunner and Bernasconi (2005) extended this line of thought, and suggested that as long as cell external sulfide (CES) concentrations are high enough, CES may diffuse back across the cytoplasmic cell membrane and may subsequently be re-oxidized to sulfate. Here, we test this hypothesis by measuring the time evolution of the δ 34S-sulfate signal during DSR in closed system experiments under different levels of sulfide stress (0–20 mM and 0–40 mM total dissolved sulfide). Our results show that the measured δ 34S-sulfate signal is markedly different in the latter case and that the observed sulfate S-isotope time-evolution is incompatible with a Rayleigh type fractionation model. In contrast, our results are consistent with a sulfate reduction and fractionation model that allows for a cell internal oxidation of dissolved sulfide by a sulfate reducer.

Single-Biomolecule Kinetics: The Art of Studying a Single Enzyme

The potential of single-enzyme studies to unravel the complex energy landscape of these polymeric catalysts is the next critical step in enzymology. From its inception in Rotman's emulsion experiments in the 1960s, the field of single-molecule enzymology has now advanced into the time-resolved age. Technological advances have enabled individual enzymatic turnover reactions to be observed with a millisecond time resolution. A number of initial studies have revealed the underlying static and dynamic disorder in the catalytic rates originating from conformational fluctuations. Although these experiments are still in their infancy, they may be able to relate the topography of the energy landscape to the biological function and regulation of enzymes. This review summarizes some of the experimental techniques and data-analysis methods that have been used to study individual enzyme molecules in search of a deeper understanding of their kinetics.

Planar silicon nitride waveguides for biosensing

The principles of attenuation of the light intensity due to multiple reflections are realised in a planar silicon oxide (SiO(2))silicon nitride (Si(3)N(4)) waveguiding structure for the purpose of developing optical biosensors with improved sensitivity. The analysis of the experimental data shows that the large difference in refractive indices of core and cladding layers gives rise to an increase in sensitivity by a factor of 3 over previously reported structures. Composite polyelectrolyte self-assembled thin films containing cyclo-tetra-chromotropylene as an indicator and enzymes glucose oxidase or urease were employed in the superstrate as a sensing membrane. Individual enzyme reactions as well as their inhibition by pesticides were studied by monitoring the intensity of light output from the planar waveguide. The results were compatible with those obtained by conventional ultraviolet-visible absorption spectroscopy. The instrument detection limit for Imidacloprid pesticide was found to be as low as 10 ppb in concentration.

Development of a kinetic model for l-lysine biosynthesis in Corynebacterium glutamicum and its application to metabolic control analysis

A mathematical model describing intracellular lysine synthesis by Corynebacterium glutamicum in batch fermentation was developed. The model is based on material balance equations of the key metabolites, and includes mechanistically based, experimentally matched rate equations for individual enzymes. From the measurements of the levels of intra- and extracellular metabolites during cultivation, the kinetic parameters in the model were identified through the decomposition of the network of reactions. The model predictions and experimental observations were in reasonable agreement. Using the model developed, metabolic control analysis was carried out to identify the rate-limiting steps, by evaluating the control on the overall lysine synthesis flux exerted by individual enzymatic reactions, which suggested how the control on lysine synthesis changes from aspartokinase to lysine permease as fermentation proceeded and indicated that lysine production could be enhanced by improving aspartokinase activity of this strain through genetic manipulation.

Identification of differentially expressed genes by restriction endonuclease-based gene expression fingerprinting.

A novel method for identification of differentially expressed genes has been developed. It is based on the consecutive restriction digestions of 3' terminal cDNA fragments to produce a fingerprint of gene expression. cDNA molecules are synthesized using a biotinylated oligo(dT) primer, digested with a frequently cutting restriction endonuclease and the 3'-terminal restriction fragments are isolated using streptavidin microbeads. After amplification by PCR, cDNA fragments are immobilized again on streptavidin beads, radiolabeled and treated sequentially with a set of restriction endonucleases. The products of individual enzymatic reactions from two or more different RNA populations are resolved by polyacrylamide gel electrophoresis and compared to reveal differentially expressed genes. This strategy enabled us to identify and clone the fragments of five genes expressed differentially in murine thymus and spleen. One of the genes was found to encode terminal deoxynucleotidyl transferase; others are apparently previously unknown genes.

Temperature and Water Status of Seed Affect Afterripening in Wild Oat (Avena fatua)

Dormant wild oat seed require afterripening under warm-dry conditions for conversion to a nondormant state capable of germination. Research was conducted to determine the relationship between temperature and seed moisture levels on afterripening of dormant wild oat line M73 seed, and to evaluate the status of water binding in the dormant seed. Conversion of dormant wild oat seed to a nondormant state at 20 to 40 C occurs primarily in the range of 5 to 20% seed moisture. There is an inverse relationship between temperature and seed moisture content for afterripening as measured by seed germination. As the afterripening temperature increases, the seed moisture content must decrease for maximum afterripening (germination) to occur. Moisture isotherms and derived enthalpy curves indicate three regions of water binding which reflect decreased binding of water to seed components as the moisture content in the dormant seed increases. Maximum afterripening, in the second region of water binding, corresponds to seed moisture contents of 7 to 22%. In this region water is weakly associated with macromolecular surfaces and begins to have solvent properties. Because afterripening occurs mainly in the second region of water binding it is likely that individual enzymatic and nonenzymatic reactions, rather than metabolic processes, mediate the conversion of wild oat seed to the nondormant state.

EAA antagonists conserve functional properties of neurons exposed to EAAS

Sterols become functional only after removal of the two methyl groups at C-4. This review focuses on the sterol C-4 demethylation process in higher plants. An intriguing aspect in the removal of the two C-4 methyl groups of sterol precursors in plants is that it does not occur consecutively as it does in yeast and animals, but is interrupted by several enzymatic steps. Each C-4 demethylation step involves the sequential participation of three individual enzymatic reactions including a sterol methyl oxidase (SMO), a 3β-hydroxysteroid-dehydrogenase/C4-decarboxylase (3βHSD/D) and a 3-ketosteroid reductase (SR). The distant location of the two C-4 demethylations in the sterol pathway requires distinct SMOs with respective substrate specificity. Combination of genetic and molecular enzymological approaches allowed a thorough identification and functional characterization of two distinct families of SMOs genes and two 3βHSD/D genes. For the latter, these studies provided the first molecularly and functionally characterized HSDs from a short chain dehydrogenase/reductase family in plants, and the first data on 3-D molecular interactions of an enzyme of the postoxidosqualene cyclase sterol biosynthetic pathway with its substrate in animals, yeast and higher plants. Characterization of these three new components involved in C-4 demethylation participates to the completion of the molecular inventory of sterol synthesis in higher plants.

Enzyme‐enzyme interactions and control analysis

Two usual assumptions of the treatment of metabolism are: (a) the rates of isolated enzyme reactions are additive, i.e., that rate is proportional to enzyme concentration; (b) in a system, the rates of individual enzyme reactions are not influenced by interactions with other enzymes, i.e. that they are acting independently, except by being coupled through shared metabolites. On this basis, control analysis has established theorems and experimental methods for studying the distribution of control. These assumptions are not universally true and it is shown that the theorems can be modified to take account of such deviations. This is achieved by defining additional elasticity coefficients, designated by the symbol pi, which quantify the effects of homologous and heterologous enzyme interactions. Here we show that for the case of non-proportionality of rate with enzyme concentration, (pi ii not equal to 1), the summation theorems are given by (Formula: see text). The example of monomer-oligomer equilibria is used to illustrate non-additive behaviour and experimental methods for their study are suggested.

Measurement of choline acetyltransferase with [14C]acetate by a cycling procedure.

A multiple enzyme and multisubstrate cycling system is described for the radiometric determination of cholineacetyltransferase (ChAT) activity in crude tissue homogenates. The methods employs [14C]acetate coupled with the enzymes acetate kinase (AK) and phosphotransacetylase (PTA) for the generation of [14C]acetyl CoA. By recycling it was possible to avoid product inhibition of ChAT by CoA, ATP was maintained constant by rephosphorylation of ADP. Kinetics of the individual enzyme reactions were studied and the parameters obtained were used to select appropriate conditions to maintain linearity of varying amounts ChAT activity over a sixty minute time course. The sensitivity of the method is limited only by the specific activity of commercially available isotope labeled acetate.

Simultaneous Determination of Three Species by Flow Injection Analytical Method Using Enzyme-Immobilized Open-Tubular Reactors

A simultaneous determination of three species by a flow injection analytical (FIA) method using enzyme-immobilized open-tubular reactors in parallel is described. The design of the optimal geometry of the FIA system is especially important to avoid overlap of any pairs of peaks. Ours was carried out based on the Taylor-Aris equation. Despite a somewhat complex splitting-confluence flow system, reproducible results were obtained in the simultaneous determination of L-lactate, β-D-glucose and glycerol by the detection of the resulting common product, NADH. Assignment of the enzyme reactors to the channels should be made by taking into account the concentration ranges of the analytes to be measured and the differences in the rates of individual enzyme reactions.

Association between the α-ketoglutarate dehydrogenase complex and succinate thiokinase

The kinetic parameters of the individual reaction of pig heart α-ketoglutarate dehydrogenase complex, succinate thiokinase and the α-ketoglutarate dehydrogenase complex-succinate thiokinase coupled system were studied. The K m CoA of α-ketoglutarate dehydrogenase complex and the K m succinyl CoA of succinate thiokinase decreased in the coupled system when compared to those of the individual enzyme reactions. This phenomenon can be explained by the interaction between the α-ketoglutarate dehydrogenase complex and succinate thiokinase. By means of poly(ethylene glycol) precipitation, ultracentrifugation and gel chromatography we were able to detect a physical interaction between the α-ketoglutarate dehydrogenase complex and succinate thiokinase. Of the seven investigated proteins only succinate thiokinase showed associated with α-ketoglutarate dehydrogenase complex. On the other hand, succinate thiokinase did not associate with other high molecular weight mitochondrial enzymes such as pyruvate dehydrogenase complex and glutamate dehydrogenase. On this basis, the interaction between succinate thiokinase and α-ketoglutarate dehydrogenase complex was assumed to be specific. These in vitro data raise the possibility that a portion of the citric acid cycle enzymes exists as a large multienzyme complex in the mitochondrial matrix.