Spectroscopic properties of oxidation species generated in the lignin of wood fibers by a laccase catalyzed treatment: electronic hole state migration and stabilization in the lignin matrix

A laccase catalyzed oxidative treatment of wood pulp fibers has been found to induce unusual modifications of these fibers that are qualitatively different from those encountered when more severely degraded fibers are subjected to similar enzymatically catalyzed oxidative treatments. These results suggest that the physical/conformational state of the lignin of wood fibers determines which oxidation pathways dominate in a given oxidative treatment, leading to different lignin modifications depending on both the chemical and the physical structure of the lignin polymer. Spectroscopic measurements (ESR, IR, UV-Vis and fluorescence) show that the laccase treatment results in the formation of two different species in the dried fibers: one is interpreted as chemically transformed (via oxygen) lignin products, and the other as initial oxidation radicals which have gained stabilization against transformation into the first mentioned products via a migration mechanism. It is argued that these initial radicals may likely be cation radical (or hole state) parts in lignin. The migration mechanism is identified with site-to-site transfer or 'hopping' via electron transfer and it is postulated that this mechanism 'carries' cation radical parts of the lignin, produced at the surface of the fiber, into parts of the lignin where chemical transformation pathways are suppressed due to the lignin conformational state. The possible existence of such a migration mechanism, the relative dominance of which should depend sensitively on the polymer conformational state, may have implications for the biogeneration and biodegradation of lignin as well as for oxidative treatments of non-natural conjugated polymers.     

Two parallel pathways in the kinetic sequence of the dihydrofolate reductase from Mycobacterium tuberculosis

Dihydrofolate reductase from Mycobacterium tuberculosis (MtDHFR) catalyzes the NAD(P)H-dependent reduction of dihydrofolate, yielding NAD(P)(+) and tetrahydrofolate, the primary one-carbon unit carrier in biology. Tetrahydrofolate needs to be recycled so that reactions involved in dTMP synthesis and purine metabolism can be maintained. Previously, steady-state studies revealed that the chemical step significantly contributes to the steady-state turnover number, but that a step after the chemical step was likely limiting the reaction rate. Here, we report the first pre-steady-state investigation of the kinetic sequence of the MtDHFR aiming to identify kinetic intermediates, and the identity of the rate-limiting steps. This kinetic analysis suggests a kinetic sequence comprising two parallel pathways with a rate-determining product release. Although product release is likely occurring in a random fashion, there is a slight preference for the release of THF first, a kinetic sequence never observed for a wild-type dihydrofolate reductase of any organism studied to date. Temperature studies were conducted to determine the magnitude of the energetic barrier posed by the chemical step, and the pH dependence of the chemical step was studied, demonstrating an acidic shift from the pK(a) observed at the steady state. The rate constants obtained here were combined with the activation energy for the chemical step to compare energy profiles for each kinetic sequence. The two parallel pathways are discussed, as well as their implications for the catalytic cycle of this enzyme.     

Molecular mechanism of the P-type ATPases

The recent determination of the structure of the Ca²⁺-ATPase of sarcoplasmic reticulum to atomic resolution in the Ca²⁺-bound state and to near atomic resolution in the Ca²⁺-free, decavanadate-bound state has paved the way for an ultimate complete understanding of the molecular mechanism of the P-type ATPases. Analysis of this new structure information together with the large amount of biochemical information about these enzymes that preceded it has produced important new revelations about how the P-type ATPases work. Most models propose that these transporters operate by a strictly conformational energy coupling mechanism in which conformational changes in the large cytoplasmic head region mechanically drive the ions to be transported from their binding sites in the transmembrane helix region 50 Å away. However, while these enzymes do indeed undergo profound conformational changes, the available evidence suggests that they do not mechanically transduce the chemical energy of ATP hydrolysis into transmembrane ion gradients via these conformational changes. As an alternative, it is proposed that the effects of the chemical events that occur at the phosphorylation/dephosphorylation site in the cytoplasmic region are exerted on the ion-binding sites via two well-defined charge transfer pathways that electronically connect the chemical reaction site with the site of ion binding. The recognition of these charge transfer pathways provides rational explanations of all of the key biochemical features of the P-type ATPase catalytic cycle. Thus, although a few details await elucidation, a nearly complete understanding of the P-type ATPase reaction mechanism may be at hand.     

Combination chemical genetics

Predicting the behavior of living organisms is an enormous challenge given their vast complexity. Efforts to model biological systems require large datasets generated by physical binding experiments and perturbation studies. Genetic perturbations have proven important and are greatly facilitated by the advent of comprehensive mutant libraries in model organisms. Small-molecule chemical perturbagens provide a complementary approach, especially for systems that lack mutant libraries, and can easily probe the function of essential genes. Though single chemical or genetic perturbations provide crucial information associating individual components (for example, genes, proteins or small molecules) with pathways or phenotypes, functional relationships between pathways and modules of components are most effectively obtained from combined perturbation experiments. Here we review the current state of and discuss some future directions for 'combination chemical genetics', the systematic application of multiple chemical or mixed chemical and genetic perturbations, both to gain insight into biological systems and to facilitate medical discoveries.     

Character coding of secondary chemical variation for use in phylogenetic analyses

A coding procedure is presented for secondary chemical data whereby putative biogenetic pathways are coded as phylogenetic characters with enzymatic conversions between compounds representing the corresponding character states. A character state tree or stepmatrix allows direct representation of the secondary chemical biogenetic pathway and avoids problems of non-independence associated with coding schemes that score presence/absence of individual compounds. Stepmatrices are the most biosynthetically realistic character definitions because individual and population level polymorphisms can be scored, reticulate enzymatic conversions within pathways may be represented, and down-weighting of pathway loss versus gain is possible. The stepmatrix approach unifies analyses of secondary chemicals, allozymes, and developmental characters because the biological unity of the pathway, locus, or character ontogeny is preserved. Empirical investigation of the stepmatrix and character state tree coding methods using floral fragrance data in Cypripedium (Orchidaceae) resulted in cladistic relationships which were largely congruent with those suggested from recent DNA and allozyme studies. This character coding methodology provides an effective means for including secondary compound data in total evidence studies. Furthermore, ancestral state reconstructions provide a phylogenetic context within which biochemical pathway evolution may be studied.     

Interpretation of nonlinear steady state enzyme kinetics—Cyclic and mathematical properties of cooperative,second-site and random pathway models

Nonlinear steady state kinetic patterns are frequently encountered in enzyme studies. Consequently, there is a need to develop procedures for systematically interpreting such data. This paper contributes to this development by identifying a common feature in nonlinear systems and by showing that quite different models commonly in use give very similar mathematical functions.Identical or similar cycles can result from quite different chemical events in enzyme mechanisms, cooperativity, second sites and random pathways. Such cycles can account for many of the observed nonlinear patterns, i.e., power functions, substrate activation and inhibition. Therefore nonlinear steady state kinetics generally requires the presence of a cycle(s) in the mechanism without specifying the underlying chemical events giving rise to that cycle(s).Rate equations for cooperative, second-site and random pathway models are derived and shown to yield virtually identical mathematical functions. Thus empirical equations composed of these functions can be used to represent nonlinear kinetic data without specifying the underlying chemical events.     

Differential native state ruggedness of the two Ca2+-binding domains in a Ca2+ sensor protein

Characterization of near-native excited states of a protein provides insights into various biological functions such as co-operativity, protein-ligand, and protein-protein interactions. In the present study, we investigated the ruggedness of the native state of EhCaBP using nonlinear temperature dependence of backbone amide-proton chemical shifts. EhCaBP is a two-domain EF-hand calcium sensor protein consisting of two EF-hands in each domain and binds four Ca2+ ions. It has been observed that approximately 30% of the residues in the protein access alternative conformations. Theoretical modeling suggested that these low-energy excited states are within 2-3 kcal/mol from the native state. Further, it is interesting to note that the residues accessing alternative conformations are more dominated in the C-terminal domain compared with its N-terminal counterpart suggesting that the former is more rugged in its native state. These distinct characteristics of N- and C-terminal domains of a calcium sensor protein belonging to the super family of calmodulin would have implications for domain dependent Ca2+ signaling pathways.     

Backbone NMR assignments of HypF-N under conditions generating toxic and non-toxic oligomers

The HypF protein is involved in the maturation and regulation of hydrogenases. The N-terminal domain of HypF (HypF-N) has served as a key model system to study the pathways of protein amyloid formation and the nature of the toxicity of pre-fibrilar protein oligomers. This domain can aggregate into two forms of oligomers having significantly different toxic effects when added to neuronal cultures. Here, NMR assignments of HypF-N backbone resonances are presented in its native state and under the conditions favouring the formation of toxic and non-toxic oligomers. The analyses of chemical shifts provide insights into the protein conformational state and the possible pathways leading to the formation of different types of oligomers.     

Open states in native polynucleotides. I. Hydrogen-exchange study of adenine-containing double helices.

Since protons that are buried and hydrogen-bonded within nucleic acid double helices exchange readily with solvent protons, it is evident that the native double helix must participate in some kind of reversible opening process. In hydrogen-exchange studies of a number of adenine-containing double helices, the chemical exchange pathways were worked out, and equilibrium and kinetic parameters of the dominant opening reactions were derived. These lead to a picture of the open state that may have implications for DNA recognition processes.     

Structural biology of the thioester-dependent degradation and synthesis of fatty acids

The fatty acid degradation and synthesis pathways consist of the same four chemical transformations. These transformations are facilitated by conjugating the fatty acid, via a thioester bond, to coenzyme A or acyl carrier protein in, respectively, the degradation and synthesis pathways. These pathways are compartmentalized in the peroxisomes, mitochondria and cytosol of eukaryotic cells. Current structural knowledge of the enzymes comprising these pathways shows that the approximately 130 entries in the RCSB Protein Data Bank can be grouped into seven superfamilies. Multifunctional enzymes are important in both pathways.     

Applications of genetically-encoded biosensors for the construction and control of biosynthetic pathways

Cells are filled with biosensors, molecular systems that measure the state of the cell and respond by regulating host processes. In much the same way that an engineer would monitor a chemical reactor, the cell uses these sensors to monitor changing intracellular environments and produce consistent behavior despite the variable environment. While natural systems derive a clear benefit from pathway regulation, past research efforts in engineering cellular metabolism have focused on introducing new pathways and removing existing pathway regulation. Synthetic biology is a rapidly growing field that focuses on the development of new tools that support the design, construction, and optimization of biological systems. Recent advances have been made in the design of genetically-encoded biosensors and the application of this class of molecular tools for optimizing and regulating heterologous pathways. Biosensors to cellular metabolites can be taken directly from natural systems, engineered from natural sensors, or constructed entirely in vitro. When linked to reporters, such as antibiotic resistance markers, these metabolite sensors can be used to report on pathway productivity, allowing high-throughput screening for pathway optimization. Future directions will focus on the application of biosensors to introduce feedback control into metabolic pathways, providing dynamic control strategies to increase the efficient use of cellular resources and pathway reliability.     

The use of microorganisms in L-ascorbic acid production

L-Ascorbic acid has been industrially produced for around 70 years. Over the past two decades, several innovative bioconversion systems have been proposed in order to simplify the long time market-dominating Reichstein method, a largely chemical synthesis by which still a considerable part of L-ascorbic acid is produced. Here, we describe the current state of biotechnological alternatives using bacteria, yeasts, and microalgae. We also discuss the potential for direct production of l-ascorbic acid exploiting novel bacterial pathways. The advantages of these novel approaches competing with current chemical and biotechnological processes are outlined.     

Insights into the chemomechanical coupling of the myosin motor from simulation of its ATP hydrolysis mechanism

The molecular motor myosin converts chemical energy from ATP hydrolysis into mechanical work, thus driving a variety of essential motility processes. Although myosin function has been studied extensively, the catalytic mechanism of ATP hydrolysis and its chemomechanical coupling to the motor cycle are not completely understood. Here, the catalysis mechanism in myosin II is examined using quantum mechanical/molecular mechanical reaction path calculations. The resulting reaction pathways, found in the catalytically competent closed/closed conformation of the Switch-1/Switch-2 loops of myosin, are all associative with a pentavalent bipyramidal oxyphosphorane transition state but can vary in the activation mechanism of the attacking water molecule and in the way the hydrogens are transferred between the heavy atoms. The coordination bond between the Mg2+ metal cofactor and Ser237 in the Switch-1 loop is broken in the product state, thereby facilitating the opening of the Switch-1 loop after hydrolysis is completed, which is required for subsequent strong rebinding to actin. This reveals a key element of the chemomechanical coupling that underlies the motor cycle, namely, the modulation of actin unbinding or binding in response to the ATP or ADP x P(i) state of nucleotide-bound myosin.     

Modeling Free Energy Availability from Hadean Hydrothermal Systems to the First Metabolism

Off-axis Hydrothermal Systems (HSs) are seen as the possible setting for the emergence of life. As the availability of free energy is a general requirement to drive any form of metabolism, we ask here under which conditions free energy generation by geologic processes is greatest and relate these to the conditions found at off-axis HSs. To do so, we present a conceptual model in which we explicitly capture the energetics of fluid motion and its interaction with exothermic reactions to maintain a state of chemical disequilibrium. Central to the interaction is the temperature at which the exothermic reactions take place. This temperature not only sets the equilibrium constant of the chemical reactions and thereby the distance of the actual state to chemical equilibrium, but these reactions also shape the temperature gradient that drives convection and thereby the advection of reactants to the reaction sites and the removal of the products that relate to geochemical free energy generation. What this conceptual model shows is that the positive feedback between convection and the chemical kinetics that is found at HSs favors a greater rate of free energy generation than in the absence of convection. Because of the lower temperatures and because the temperature of reactions is determined more strongly by these dynamics rather than an external heat flux, the conditions found at off-axis HSs should result in the greatest rates of geochemical free energy generation. Hence, we hypothesize from these thermodynamic considerations that off-axis HSs seem most conducive for the emergence of protometabolic pathways as these provide the greatest, abiotic generation rates of chemical free energy.     

Network methods for describing sample relationships in genomic datasets: application to Huntington’s disease

Background

We consider the possibility of engineering metabolic pathways in a chassis organism in order to synthesize novel target compounds that are heterologous to the chassis. For this purpose, we model metabolic networks through hypergraphs where reactions are represented by hyperarcs. Each hyperarc represents an enzyme-catalyzed reaction that transforms set of substrates compounds into product compounds. We follow a retrosynthetic approach in order to search in the metabolic space (hypergraphs) for pathways (hyperpaths) linking the target compounds to a source set of compounds.

Results

To select the best pathways to engineer, we have developed an objective function that computes the cost of inserting a heterologous pathway in a given chassis organism. In order to find minimum-cost pathways, we propose in this paper two methods based on steady state analysis and network topology that are to the best of our knowledge, the first to enumerate all possible heterologous pathways linking a target compounds to a source set of compounds. In the context of metabolic engineering, the source set is composed of all naturally produced chassis compounds (endogenuous chassis metabolites) and the target set can be any compound of the chemical space. We also provide an algorithm for identifying precursors which can be supplied to the growth media in order to increase the number of ways to synthesize specific target compounds.

Conclusions

We find the topological approach to be faster by several orders of magnitude than the steady state approach. Yet both methods are generally scalable in time with the number of pathways in the metabolic network. Therefore this work provides a powerful tool for pathway enumeration with direct application to biosynthetic pathway design.     

Exploring the diversity of complex metabolic networks

MOTIVATION: Metabolism, the network of chemical reactions that make life possible, is one of the most complex processes in nature. We describe here the development of a computational approach for the identification of every possible biochemical reaction from a given set of enzyme reaction rules that allows the de novo synthesis of metabolic pathways composed of these reactions, and the evaluation of these novel pathways with respect to their thermodynamic properties. RESULTS: We applied this framework to the analysis of the aromatic amino acid pathways and discovered almost 75,000 novel biochemical routes from chorismate to phenylalanine, more than 350,000 from chorismate to tyrosine, but only 13 from chorismate to tryptophan. Thermodynamic analysis of these pathways suggests that the native pathways are thermodynamically more favorable than the alternative possible pathways. The pathways generated involve compounds that exist in biological databases, as well as compounds that exist in chemical databases and novel compounds, suggesting novel biochemical routes for these compounds and the existence of biochemical compounds that remain to be discovered or synthesized through enzyme and pathway engineering. AVAILABILITY: Framework will be available via web interface at http://systemsbiology.northwestern.edu/BNICE (site under construction). CONTACT: vassily@northwestern.edu or broadbelt@northwestern.edu SUPPLEMENTARY INFORMATION: http://systemsbiology.northwestern.edu/BNICE/publications.     

Regulators of cardiac fibroblast cell state

Fibroblasts are the primary regulator of cardiac extracellular matrix (ECM). In response to disease stimuli cardiac fibroblasts undergo cell state transitions to a myofibroblast phenotype, which underlies the fibrotic response in the heart and other organs. Identifying regulators of fibroblast state transitions would inform which pathways could be therapeutically modulated to tactically control maladaptive extracellular matrix remodeling. Indeed, a deeper understanding of fibroblast cell state and plasticity is necessary for controlling its fate for therapeutic benefit. p38 mitogen activated protein kinase (MAPK), which is part of the noncanonical transforming growth factor β (TGFβ) pathway, is a central regulator of fibroblast to myofibroblast cell state transitions that is activated by chemical and mechanical stress signals. Fibroblast intrinsic signaling, local and global cardiac mechanics, and multicellular interactions individually and synergistically impact these state transitions and hence the ECM, which will be reviewed here in the context of cardiac fibrosis.     

Deactivation mechanisms of triplet excited state hypericin by β-carotene

The deactivation mechanisms of the triplet excited state hypericin (HYP) by β-carotene (CAR) were studied employing quantum chemical calculations in the present study. The results suggest that CAR may deactivate the triplet excited state HYP through the following two pathways on thermodynamic grounds: (1) direct energy transfer from the triplet excited state HYP to CAR; (2) electron transfer from the triplet excited state CAR, which was formed through direct energy transfer pathway, to the triplet excited state HYP.     

The chemical organization of signaling interactions

MOTIVATION: Cellular chemical signaling pathways form complex networks that are beginning to be studied at the level of chemical kinetics and databases of reactions. Chemical reaction details are traditionally represented as lists of reactions and rates. This does not map readily to the block diagram representation familiar to biologists, and obscures the functional organization of signaling networks. This study examines motifs in signaling chemistry and reports common features that may help to formalize such a mapping between pathway block diagrams and the chemistry. The same motifs may facilitate data representation and provide functional abstraction of the chemistry. RESULTS: I classified 74 interactions between 25 signaling pathways in terms of shared chemical motifs. All interactions in this dataset consist of a few communicating molecules from one set of pathways, and a replicating set of reactions and molecules from another. Each unique combination of interacting pathways duplicates the chemical reaction scheme of this replicating set, but involves different rate constants. Signaling pathways can therefore be described in an object-oriented manner as sets of core reactions with well-defined interfaces between pathways. This generalization lends itself to designing simulators and databases for signaling networks. AVAILABILITY: Software and example models are freely available from http://www.ncbs.res.in/~bhalla/examples/EGFR_example.html.