Channeling in native microbial pathways: Implications and challenges for metabolic engineering

Intracellular enzymes can be organized into a variety of assemblies, shuttling intermediates from one active site to the next. Eukaryotic compartmentalization within mitochondria and peroxisomes and substrate tunneling within multi-enzyme complexes have been well recognized. Intriguingly, the central pathways in prokaryotes may also form extensive channels, including the heavily branched glycolysis pathway. In vivo channeling through cascade enzymes is difficult to directly measure, but can be inferred from in vitro tests, reaction thermodynamics, transport/reaction modeling, analysis of molecular diffusion and protein interactions, or steady state/dynamic isotopic labeling. Channeling presents challenges but also opportunities for metabolic engineering applications. It rigidifies fluxes in native pathways by trapping or excluding metabolites for bioconversions, causing substrate catabolite repressions or inferior efficiencies in engineered pathways. Channeling is an overlooked regulatory mechanism used to control flux responses under environmental/genetic perturbations. The heterogeneous distribution of intracellular enzymes also confounds kinetic modeling and multiple-omics analyses. Understanding the scope and mechanisms of channeling in central pathways may improve our interpretation of robust fluxomic topology throughout metabolic networks and lead to better design and engineering of heterologous pathways.     

Chloroplasts as source and target of cellular redox regulation: a discussion on chloroplast redox signals in the context of plant physiology

During the evolution of plants, chloroplasts have lost the exclusive genetic control over redox regulation and antioxidant gene expression. Together with many other genes, all genes encoding antioxidant enzymes and enzymes involved in the biosynthesis of low molecular weight antioxidants were transferred to the nucleus. On the other hand, photosynthesis bears a high risk for photo-oxidative damage. Concomitantly, an intricate network for mutual regulation by anthero- and retrograde signals has emerged to co-ordinate the activities of the different genetic and metabolic compartments. A major focus of recent research in chloroplast regulation addressed the mechanisms of redox sensing and signal transmission, the identification of regulatory targets, and the understanding of adaptation mechanisms. In addition to redox signals communicated through signalling cascades also used in pathogen and wounding responses, specific chloroplast signals control nuclear gene expression. Signalling pathways are triggered by the redox state of the plastoquinone pool, the thioredoxin system, and the acceptor availability at photosystem I, in addition to control by oxolipins, tetrapyrroles, carbohydrates, and abscisic acid. The signalling function is discussed in the context of regulatory circuitries that control the expression of antioxidant enzymes and redox modulators, demonstrating the principal role of chloroplasts as the source and target of redox regulation.     

Conversion of the noncooperative Bacillus subtilis aspartate transcarbamoylase into a cooperative enzyme by a single amino acid substitution.

Allosteric enzymes are part of a unique class of enzymes which regulate metabolic pathways. On the molecular level, allosteric regulation is the result of interactions between discrete binding sites on the enzyme. In order to accommodate these multiple binding sites, allosteric enzymes have evolved with oligomeric quaternary structures. However, only a few oligomeric enzymes are known to have regulatory interactions between binding sites. Is regulatory activity an inherent property of oligomeric enzymes? The trimeric Bacillus subtilis aspartate transcarbamoylase catalyzes the first committed step of the pyrimidine biosynthetic pathway and is not known to be a regulatory enzyme. When an alanine residue is substituted for the active-site residue Arg-99 by site-specific mutagenesis, the regulatory activity of homotropic substrate cooperativity (Hill coefficient of 1.5) is observed in the resulting mutant enzyme. These results suggest that homotropic regulation may have evolved by a relatively small number of mutations to an oligomeric enzyme.     

Streptomyces sp. is a powerful biotechnological tool for the biodegradation of HCH isomers: biochemical and molecular basis

Actinobacteria are well-known degraders of toxic materials that have the ability to tolerate and remove organochloride pesticides; thus, they are used for bioremediation. The biodegradation of organochlorines by actinobacteria has been demonstrated in pure and mixed cultures with the concomitant production of metabolic intermediates including γ-pentachlorocyclohexene (γ-PCCH); 1,3,4,6-tetrachloro-1,4-cyclohexadiene (1,4-TCDN); 1,2-dichlorobenzene (1,2-DCB), 1,3-dichlorobenzene (1,3-DCB), or 1,4-dichlorobenzene (1,4-DCB); 1,2,3-trichlorobenzene (1,2,3-TCB), 1,2,4-trichlorobenzene (1,2,4-TCB), or 1,3,5-trichlorobenzene (1,3,5-TCB); 1,3-DCB; and 1,2-DCB. Chromatography coupled to mass spectrometric detection, especially GC–MS, is typically used to determine HCH-isomer metabolites. The important enzymes involved in HCH isomer degradation metabolic pathways include hexachlorocyclohexane dehydrochlorinase (LinA), haloalkane dehalogenase (LinB), and alcohol dehydrogenase (LinC). The metabolic versatility of these enzymes is known. Advances have been made in the identification of actinobacterial haloalkane dehydrogenase, which is encoded by linB. This knowledge will permit future improvements in biodegradation processes using Actinobacteria. The enzymatic and genetic characterizations of the molecular mechanisms involved in these processes have not been fully elucidated, necessitating further studies. New advances in this area suggest promising results. The scope of this paper encompasses the following: (i) the aerobic degradation pathways of hexachlorocyclohexane (HCH) isomers; (ii) the important genes and enzymes involved in the metabolic pathways of HCH isomer degradation; and (iii) the identification and quantification of intermediate metabolites through gas chromatography coupled to mass spectrometry (GC–MS).     

Metabolic enzymes that bind RNA: yet another level of cellular regulatory network?

Several enzymes that were originally characterized to have one defined function in intermediatory metabolism are now shown to participate in a number of other cellular processes. Multifunctional proteins may be crucial for building of the highly complex networks that maintain the function and structure in the eukaryotic cell possessing a relatively low number of protein-encoding genes. One facet of this phenomenon, on which I will focus in this review, is the interaction of metabolic enzymes with RNA. The list of such enzymes known to be associated with RNA is constantly expanding, but the most intriguing question remains unanswered: are the metabolic enzyme-RNA interactions relevant in the regulation of cell metabolism? It has been proposed that metabolic RNA-binding enzymes participate in general regulatory circuits linking a metabolic function to a regulatory mechanism, similar to the situation of the metabolic enzyme aconitase, which also functions as iron-responsive RNA-binding regulatory element. However, some authors have cautioned that some of such enzymes may merely represent "molecular fossils" of the transition from an RNA to a protein world and that the RNA-binding properties may not have a functional significance. Here I will describe enzymes that have been shown to interact with RNA (in several cases a newly discovered RNA-binding protein has been identified as a well-known metabolic enzyme) and particularly point out those whose ability to interact with RNA seems to have a proven physiological significance. I will also try to depict the molecular switch between an enzyme's metabolic and regulatory functions in cases where such a mechanism has been elucidated. For most of these enzymes relations between their enzymatic functions and RNA metabolism are unclear or seem not to exist. All these enzymes are ancient, as judged by their wide distribution, and participate in fundamental biochemical pathways.     

Stress eating and tuning out: Cancer cells re-wire metabolism to counter stress

Abstract

Cancer cells reprogram metabolism to maintain rapid proliferation under often stressful conditions. Glycolysis and glutaminolysis are two central pathways that fuel cancer metabolism. Allosteric regulation and metabolite driven post-translational modifications of key metabolic enzymes allow cancer cells glycolysis and glutaminolysis to respond to changes in nutrient availability and the tumor microenvironment. While increased aerobic glycolysis (the Warburg effect) has been a noted part of cancer metabolism for over 80 years, recent work has shown that the elevated levels of glycolytic intermediates are critical to cancer growth and metabolism due to their ability to feed into the anabolic pathways branching off glycolysis such as the pentose phosphate pathway and serine biosynthesis pathway. The key glycolytic enzymes phosphofructokinase-1 (PFK1), pyruvate kinase (PKM2) and phosphoglycerate mutase 1 (PGAM1) are regulated by upstream and downstream metabolites to balance glycolytic flux with flux through anabolic pathways. Glutamine regulation is tightly controlled by metabolic intermediates that allosterically inhibit and activate glutamate dehydrogenase, which fuels the tricarboxylic acid cycle by converting glutamine derived glutamate to α-ketoglutarate. The elucidation of these key allosteric regulatory hubs in cancer metabolism will be essential for understanding and predicting how cancer cells will respond to drugs that target metabolism. Additionally, identification of the structures involved in allosteric regulation will inform the design of anti-metabolism drugs which bypass the off-target effects of substrate mimics. Hence, this review aims to provide an overview of allosteric control of glycolysis and glutaminolysis.     

Biochemical pathways and mechanisms nitrogen, amino acid, and carbon metabolism

For both nitrogen and carbon metabolism there exist specific regulatory mechanisms to enable cells to assimilate a wide variety of nitrogen and carbon sources. Superimposed are regulatory circuits, the so called nitrogen and carbon catabolite regulation, to allow for selective use of “rich” sources first and “poor” sources later. Evidence points to the importance of specific regulatory mechanisms for short term adaptations, while generalized control circuits are used for long term modulation of nitrogen and carbon metabolism. Similarly a variety of regulatory mechanisms operate in amino acid metabolism. Modulation of enzyme activity and modulation of enzyme levels are the outstanding regulatory mechanisms. In prokaryotes, attenuation and repressor/operator control are predominant, besides a so called “metabolic control” which integrates amino acid metabolism into the overall nutritional status of the cells. In eukaryotic cells compartmentation of amino acid metabolites as well as of part of the pathways becomes an additional regulatory factor; pathway specific controls seem to be rare, but a complex regulatory network, the “general control of amino acid biosynthesis”, coordinates the synthesis of enzymes of a number of amino acid biosynthetic pathways.     

Compartmentation in plant metabolism

Cell fractionation and immunohistochemical studies in the last 40 years have revealed the extensive compartmentation of plant metabolism. In recent years, new protein mass spectrometry and fluorescent-protein tagging technologies have accelerated the flow of information, especially for Arabidopsis thaliana, but the intracellular locations of the majority of proteins in the plant proteome are still not known. Prediction programs that search for targeting information within protein sequences can be applied to whole proteomes, but predictions from different programs often do not agree with each other or, indeed, with experimentally determined results. The compartmentation of most pathways of primary metabolism is generally covered in plant physiology textbooks, so the focus here is mainly on newly discovered metabolic pathways in plants or pathways that have recently been revised. Ultimately, all of the pathways of plant metabolism are interconnected, and a major challenge facing plant biochemists is to understand the regulation and control of metabolic networks. One of the best-characterized networks links sucrose synthesis in the cytosol with photosynthetic CO(2) fixation and starch synthesis in the chloroplasts. One of the key features of this network is how the transport of pathway intermediates and signal metabolites across the chloroplast envelope conveys information between the two compartments, influencing the regulation of several enzymes to co-ordinate fluxes through the different pathways. It is widely accepted that chloroplasts and mitochondria originated from prokaryotic endosymbionts, and that new transporters and regulatory networks evolved to integrate metabolism in these organelles with the rest of the cell. Curiously, the present-day locations of many metabolic pathways within the cell often do not reflect their evolutionary origin, and there is evidence of extensive shuffling of enzymes and whole pathways between compartments during the evolution of plants.     

Structural fingerprints,interactions, and signaling networks of RAS family proteins beyond RAS isoforms

Among the signaling molecules indirectly linked to many different cell surface receptors, RAS proteins essentially respond to a diverse range of extracellular cues. They control activities of multiple signaling pathways and consequently a wide array of cellular processes, including survival, growth, adhesion, migration, and differentiation. Any dysregulation of these pathway leads, thus, to cancer, developmental disorders, metabolic, and cardiovascular diseases. The biochemistry of RAS family proteins has become multifaceted since the discovery of the first members, more than 40 years ago. Substantial knowledge has been attained about molecular mechanisms underlying post-translational modification, membrane localization, regulation, and signal transduction through diverse effector molecules. However, the increasing complexity of the underlying signaling mechanisms is considerable, in part due to multiple effector pathways, crosstalks between them and eventually feedback mechanisms. Here, we take a broad view of regulatory and signaling networks of all RAS family proteins that extends beyond RAS paralogs. As described in this review, a lot is known but a lot has to be discovered yet.     

Plant secondary metabolites and vertebrate herbivores--from physiological regulation to ecosystem function

Plant secondary metabolites can constrain the diet of vertebrates and these effects can flow through to community dynamics. Recent studies have moved beyond attempting to correlate diet choice with secondary metabolite profiles and instead focus on mechanisms that animals use to detect toxins and to regulate their intake and absorption. These include molecularly determined taste specificity, serotonin-mediated learning and the control of toxin absorption by permeability-glycoproteins. Focus on the detoxification pathways employed by specialist and generalist herbivores has facilitated explicit tests of the long-standing hypothesis that detoxification rates limit feeding. Understanding the molecular basis of differences amongst species in their tolerance of plant secondary metabolites opens many opportunities for understanding the evolutionary history of interactions between vertebrates and their food plants.     

Metabolomics in systems microbiology

Because of the importance of microbes as model organisms, biotechnology tools, and contributors to mammalian and ecosystem metabolism, there has been longstanding interest in measuring their metabolite levels. Current metabolomic methods, involving mass spectrometry-based measurement of cell extracts, enable routine quantitation of most central metabolites. Metabolomics alone, however, is inadequate to understand cellular metabolic activity: Flux measurement and proteomic, genetic, and biochemical approaches with a metabolomics bent are all needed. Here we highlight examples where these integrated methods have contributed to discovery of metabolic pathways, regulatory interactions, and homeostasis mechanisms. We also indicate enduring challenges concerning unstable and low abundance compounds, subcellular compartmentalization, and quantitative amalgamation of different data types.     

Metabolic rate depression and biochemical adaptation in anaerobiosis, hibernation and estivation

For many animals, the best defense against harsh environmental conditions is an escape to a hypometabolic or dormant state. Facultative metabolic rate depression is the common adaptive strategy of anaerobiosis, hibernation, and estivation, as well as a number of other arrested states. By reducing metabolic rate by a factor ranging from 5 to 100 fold or more, animals gain a comparable extension of survival time that can support months or even years of dormancy. The present review focuses on the molecular control mechanisms that regulate and coordinate cellular metabolism for the transition into dormancy. These include reversible control over the activity state of enzymes via protein phosphorylation or dephosphorylation reactions, pathway regulation via the association or dissociation of particle-bound enzyme complexes, and fructose-2,6-bisphosphate regulation of the use of carbohydrate reserves for biosynthetic purposes. These mechanisms, their interactions, and the regulatory signals (e.g., second messenger molecules, pH) that coordinate them form a common molecular basis for metabolic depression in anoxia-tolerant vertebrates (goldfish, turtles) and invertebrates (marine molluscs), hibernation in small mammals, and estivation in land snails and terrestrial toads.     

Constructing de novo biosynthetic pathways for chemical synthesis inside living cells

Living organisms have evolved a vast array of catalytic functions that make them ideally suited for the production of medicinally and industrially relevant small-molecule targets. Indeed, native metabolic pathways in microbial hosts have long been exploited and optimized for the scalable production of both fine and commodity chemicals. Our increasing capacity for DNA sequencing and synthesis has revealed the molecular basis for the biosynthesis of a variety of complex and useful metabolites and allows the de novo construction of novel metabolic pathways for the production of new and exotic molecular targets in genetically tractable microbes. However, the development of commercially viable processes for these engineered pathways is currently limited by our ability to quickly identify or engineer enzymes with the correct reaction and substrate selectivity as well as the speed by which metabolic bottlenecks can be determined and corrected. Efforts to understand the relationship among sequence, structure, and function in the basic biochemical sciences can advance these goals for synthetic biology applications while also serving as an experimental platform for elucidating the in vivo specificity and function of enzymes and reconstituting complex biochemical traits for study in a living model organism. Furthermore, the continuing discovery of natural mechanisms for the regulation of metabolic pathways has revealed new principles for the design of high-flux pathways with minimized metabolic burden and has inspired the development of new tools and approaches to engineering synthetic pathways in microbial hosts for chemical production.     

Early Steps in Isoprenoid Biosynthesis: Multilevel Regulation of the Supply of Common Precursors in Plant Cells

Isoprenoids are produced in all organisms but are especially abundant and diverse in plants. Two separate pathways operate in plant cells to synthesize prenyl diphosphate precursors common to all isoprenoids. Cytosolic and mitochondrial precursors are produced by the mevalonic acid (MVA) pathway whereas the recently discovered methylerythritol phosphate (MEP) pathway is located in plastids. However, both pathways may participate in the synthesis of at least some isoprenoids under certain circumstances. Although genes encoding all the enzymes from both pathways have already been cloned, little is known about the regulatory mechanisms that control the supply of isoprenoid precursors. Genetic approaches are providing valuable information on the regulation of both pathways. Thus, recent data from overexpression experiments in transgenic plants show that several enzymes share control over the metabolic flux through the MEP pathway, whereas a single regulatory step has been proposed for the MVA pathway. Identification of Arabidopsis thaliana mutants that are resistant to the inhibition of the MVA and the MEP pathways is a promising approach to uncover mechanisms involved in the crosstalk between pathways. The characterization of some of these mutants impaired in light perception and signaling has recently provided genetic evidence for a role of light as a key factor to modulate the availability of isoprenoid precursors in Arabidopsis seedlings. The picture emerging from recent data supports that a complex regulatory network appears to be at work in plant cells to ensure the supply of isoprenoid precursors when needed.     

bZIP转录因子调控植物次生代谢产物生物合成的研究进展

植物次生代谢产物是通过次生代谢产生的一类小分子有机化合物,是植物适应环境的表现,次生代谢产物也是重要药物和化工原料的来源。bZIP转录因子是普遍存在于真核生物中的一类多基因家族,可有效调控植物次生代谢产物的生物合成。本文概述了植物bZIP转录因子的结构和类型,重点阐述了bZIP转录因子调控萜类、黄酮类和生物碱等植物次生代谢产物生物合成的研究进展,并对研究前景进行了展望。深入探讨bZIP转录因子的调控机制,有助于利用基因工程技术优化植物次生代谢途径,提高次生代谢产物的含量,在新药创制、工农业生产等方面具有广泛的应用前景。