Evidence for enzyme complexes in the phenylpropanoid and flavonoid pathways

A growing body of evidence indicates that phenylpropanoid and flavonoid metabolism is catalyzed, not by free-floating 'soluble' enzymes, but via one or more membrane-associated multienzyme complexes. This type of macromolecular organization has important implications for the overall efficiency, specificity, and regulation of these pathways. Classical biochemical studies of phenylpropanoid and flavonoid metabolism have laid a solid foundation for this model, providing evidence of the channeling of intermediates between enzyme active sites and co-localization of enzymes in cell membranes. This work is now being extended using transgenic plants to determine how the partitioning of metabolites within these pathways is controlled, as well as applying sensitive methods to define specific interactions among the individual enzymes. Information from these studies promises to provide new insights into the structuring of biosynthetic pathways within cells, which should lead to more effective means for engineering the production of plant metabolites with nutritional and agronomic importance.     

Evolution of enzyme structure.

Three-dimensional structures of enzymes offer evidence about their evolution. There are clear examples of divergent families (e.g. mammalian serine proteases) and convergence (e.g. chymotrypsin and subtilisin). Topological similarities in dehydrogenases may reflect an ancient divergence or merely chemical constraints on protein architectures. Further experimental evidence is desirable to back up arguments based on molecular morphology. By growing microorganisms on novel foodstuffs in a chemostat, one can focus selective pressure on a specific enzyme activity. Experiments will be described in which such pressure is focused on pentitol metabolism. Examination of the fine structure of the genes responsible for this pentitol metabolism has given clues about the volution of metabolic pathways.     

The University of Minnesota Biocatalysis/Biodegradation Database: emphasizing enzymes

The University of Minnesota Biocatalysis/Biodegradation Database (UM-BBD, http://umbbd.ahc.umn.edu/) provides curated information on microbial catabolic enzymes and their organization into metabolic pathways. Currently, it contains information on over 400 enzymes. In the last year the enzyme page was enhanced to contain more internal and external links; it also displays the different metabolic pathways in which each enzyme participates. In collaboration with the Nomenclature Commission of the International Union of Biochemistry and Molecular Biology, 35 UM-BBD enzymes were assigned complete EC codes during 2000. Bacterial oxygenases are heavily represented in the UM-BBD; they are known to have broad substrate specificity. A compilation of known reactions of naphthalene and toluene dioxygenases were recently added to the UM-BBD; 73 and 108 were listed respectively. In 2000 the UM-BBD is mirrored by two prestigious groups: the European Bioinformatics Institute and KEGG (the Kyoto Encyclopedia of Genes and Genomes). Collaborations with other groups are being developed. The increased emphasis on UM-BBD enzymes is important for predicting novel metabolic pathways that might exist in nature or could be engineered. It also is important for current efforts in microbial genome annotation.     

The metabolic organization of a primitive air-breathing fish, the Florida gar (Lepisosteus platyrhincus)

The metabolic organization of the air-breathing Florida gar, Lepisosteus platyrhincus, was assessed by measuring the maximal activities of key enzymes in several metabolic pathways in selected tissues, concentrations of plasma metabolites including nonesterified fatty acids (NEFA), free amino acids (FAA) and glucose as well as tissue FAA levels. In general, L. platyrhincus has an enhanced capacity for carbohydrate metabolism as indicated by elevated plasma glucose levels and high activities of gluconeogenic and glycolytic enzymes. Based upon these properties, glucose appears to function as the major fuel source in the Florida gar. The capacity for lipid metabolism in L. platyrhincus appears limited as plasma NEFA levels and the activities of enzymes involved in lipid oxidation are low relative to many other fish species. L. platyrhincus is capable of oxidizing both D- and L-beta-hydroxybutyrate, with tissue-specific preferences for each stereoisomer, yet the capacity for ketone body metabolism is low compared with other primitive fishes. Based on enzyme activities, the metabolism of the air-breathing organ more closely resembles that of the mammalian lung than a fish swim bladder. The Florida gar sits phylogenetically and metabolically in an intermediate position between the "primitive" elasmobranchs and the "advanced" teleosts. The apparently unique metabolic organization of the gar may have evolved in the context of a bimodal air-breathing environmental adaptation.     

MetaCyc and AraCyc. Metabolic pathway databases for plant research

MetaCyc (http://metacyc.org) contains experimentally determined biochemical pathways to be used as a reference database for metabolism. In conjunction with the Pathway Tools software, MetaCyc can be used to computationally predict the metabolic pathway complement of an annotated genome. To increase the breadth of pathways and enzymes, more than 60 plant-specific pathways have been added or updated in MetaCyc recently. In contrast to MetaCyc, which contains metabolic data for a wide range of organisms, AraCyc is a species-specific database containing only enzymes and pathways found in the model plant Arabidopsis (Arabidopsis thaliana). AraCyc (http://arabidopsis.org/tools/aracyc/) was the first computationally predicted plant metabolism database derived from MetaCyc. Since its initial computational build, AraCyc has been under continued curation to enhance data quality and to increase breadth of pathway coverage. Twenty-eight pathways have been manually curated from the literature recently. Pathway predictions in AraCyc have also been recently updated with the latest functional annotations of Arabidopsis genes that use controlled vocabulary and literature evidence. AraCyc currently features 1,418 unique genes mapped onto 204 pathways with 1,156 literature citations. The Omics Viewer, a user data visualization and analysis tool, allows a list of genes, enzymes, or metabolites with experimental values to be painted on a diagram of the full pathway map of AraCyc. Other recent enhancements to both MetaCyc and AraCyc include implementation of an evidence ontology, which has been used to provide information on data quality, expansion of the secondary metabolism node of the pathway ontology to accommodate curation of secondary metabolic pathways, and enhancement of the cellular component ontology for storing and displaying enzyme and pathway locations within subcellular compartments.     

Evolution of enzymes in metabolism: a network perspective

Several models have been proposed to explain the origin and evolution of enzymes in metabolic pathways. Initially, the retro-evolution model proposed that, as enzymes at the end of pathways depleted their substrates in the primordial soup, there was a pressure for earlier enzymes in pathways to be created, using the later ones as initial template, in order to replenish the pools of depleted metabolites. Later, the recruitment model proposed that initial templates from other pathways could be used as long as those enzymes were similar in chemistry or substrate specificity. These two models have dominated recent studies of enzyme evolution. These studies are constrained by either the small scale of the study or the artificial restrictions imposed by pathway definitions. Here, a network approach is used to study enzyme evolution in fully sequenced genomes, thus removing both constraints. We find that homologous pairs of enzymes are roughly twice as likely to have evolved from enzymes that are less than three steps away from each other in the reaction network than pairs of non-homologous enzymes. These results, together with the conservation of the type of chemical reaction catalyzed by evolutionarily related enzymes, suggest that functional blocks of similar chemistry have evolved within metabolic networks. One possible explanation for these observations is that this local evolution phenomenon is likely to cause less global physiological disruptions in metabolism than evolution of enzymes from other enzymes that are distant from them in the metabolic network.     

Organization of enzymes on subcellular structures: relay at the surface

There is a large body of evidence that soluble cytoplasmic enzymes of eukaryotic cells, e.g., glycolytic enzymes and proteins of the translational machinery, are organized in some way in space and in time. The following features of such organization emerge from the experimental data: (1) metabolites are transferred between enzymes directly "from hand to hand" in short-living enzyme-enzyme complexes rather than by diffusion in aqueous media; (2) enzymes show a tendency to be absorbed on surfaces of subcellular structures, such as membranes, cytoskeleton and polyribosomes; (3) enzymes are desorbed from a surface of a subcellular structure after binding specific metabolites, i.e., substrates and/or products of the reactions catalyzed by these enzymes. These features are suggestive of a relay mechanism for the enzyme systems functioning in a cell; an enzyme adsorbed on a surface of a subcellular structure is desorbed after binding its substrate or in the course of the catalytic act. Within a complex with its product the enzyme diffuses into the environment, until it reaches the next enzyme adsorbed on the same surface; then a short-living enzyme-enzyme complex is formed, and a direct "from hand to hand" transfer of the metabolite takes place. As a result, the overall metabolic process appears to be localized near the surface. We termed this mechanism as a "relay at the surface".     

Metabolons involving plant cytochrome P450s

Arranging biological processes into “compartments” is a key feature of all eukaryotic cells. Through this mechanism, cells can drastically increase metabolic efficiency and manage complex cellular processes more efficiently, saving space and energy. Compartmentation at the molecular level is mediated by metabolons. A metabolon is an ordered protein complex of sequential metabolic enzymes and associated cellular structural elements. The sub-cellular organization of enzymes involved in the synthesis and storage of plant natural products appears to involve the anchoring of metabolons by cytochrome P450 monooxygenases (P450s) to specific domains of the endoplasmic reticulum (ER) membrane. This review focuses on the current evidence supporting the organization of metabolons around P450s on the surface of the ER. We␣outline direct and indirect experimental data that describes P450 enzymes in the phenylpropanoid, flavonoid, cyanogenic glucoside, and other biosynthetic pathways. We also discuss the limitations and future directions of metabolon research and the potential for application to metabolic engineering endeavors.     

Reversible acetylation of metabolic enzymes celebration: SIRT2 and p300 join the party

Compelling evidence suggests that metabolic pathways are coordinated through reversible acetylation of metabolic enzymes in response to nutrient availability. In this issue of Molecular Cell, Jiang et al. (2011) show that the rate-limiting enzyme in gluconeogenesis, phosphoenolpyruvate carboxykinase 1, is regulated through reversible acetylation by SIRT2 and p300.     

On the origin of intracellular compartmentation and organized metabolic systems

The history of the development of the ideas and research of organized metabolic systems during last three decades is shortly reviewed. The cell cytoplasm is crowded with solutes, soluble macromolecules such as enzymes, nucleic acids, structural proteins and membranes. The high protein density within the large compartments of the cells predominantly determines the major characteristics of cellular environment such as viscosity, diffusion and inhomogeneity. The fact that the solvent viscosity of cytoplasm is not substantially different from the water is explained by intracellular structural heterogeneity: the intrinsic macromolecular density is relatively low within the interstitial voids in the cell because many soluble enzymes are apparently integral parts of the insoluble cytomatrix and are not distributed homogeneously. The molecular crowding and sieving restrict the mobility of very large solutes, binding severely restrict the mobility of smaller solutes. One of consequence of molecular crowding and hindered diffusion is the need to compartmentalize metabolic pathway to overcome diffusive barriers. Although the movement of small molecules is slowed down in the cytoplasm, the metabolism can successfully proceed and even be facilitated by metabolite channeling which directly transfers the intermediate from one enzyme to an adjacent enzyme without the need of free aqueous-phase diffusion. The enhanced probability for intermediates to be transferred from one active site to the other by sequential enzymes requires stable or transient interactions of the relevant enzymes, which associate physically in non-dissociable, static multienzyme complexes--metabolones, particles containing enzymes of a part or whole metabolic systems. Therefore, within the living cell the metabolism depends on the structural organization of enzymes forming microcompartments. Since cells contain many compartments and microenvironments, the measurement of the concentration of metabolites in whole cells or tissues gives an average cellular concentration and not that which is actually sensed by the active site of a specific enzyme. Thus, the microcompartmentation could provide a mechanism which can control metabolic pathways. Independently and in parallel to the developments described above, the ideas of compartmentation came into existence from the necessity to explain important physiological phenomena, in particular in heart research and in cardiac electrophysiology. These phenomena demonstrated the physiological importance of the biophysical and biochemical mechanisms described in this review.     

Enzyme-specific profiles for genome annotation: PRIAM

The advent of fully sequenced genomes opens the ground for the reconstruction of metabolic pathways on the basis of the identification of enzyme-coding genes. Here we describe PRIAM, a method for automated enzyme detection in a fully sequenced genome, based on the classification of enzymes in the ENZYME database. PRIAM relies on sets of position-specific scoring matrices ('profiles') automatically tailored for each ENZYME entry. Automatically generated logical rules define which of these profiles is required in order to infer the presence of the corresponding enzyme in an organism. As an example, PRIAM was applied to identify potential metabolic pathways from the complete genome of the nitrogen-fixing bacterium Sinorhizobium meliloti. The results of this automated method were compared with the original genome annotation and visualised on KEGG graphs in order to facilitate the interpretation of metabolic pathways and to highlight potentially missing enzymes.     

Metabolic organization of the spotted ratfish, Hydrolagus colliei (Holocephali: Chimaeriformes): insight into the evolution of energy metabolism in the chondrichthyan fishes

The metabolic organization of a holocephalan, the spotted ratfish (Hydrolagus colliei), was assessed using measurements of key enzymes of several metabolic pathways in four tissues and plasma concentrations of free amino acids (FAA) and non-esterified fatty acids (NEFA) to ascertain if the Holocephali differ metabolically from the Elasmobranchii since these groups diverged ca. 400 Mya. Activities of carnitine palmitoyl transferase indicate that fatty acid oxidation occurs in liver and kidney but not in heart or white muscle. This result mirrors the well-established absence of lipid oxidation in elasmobranch muscle, and more recent studies showing that elasmobranch kidney possesses a capacity for lipid oxidation. High activities in oxidative tissues of enzymes of ketone body metabolism, including D-beta-hydroxybutyrate dehydrogenase, indicate that, like elasmobranchs, ketone bodies are of central importance in spotted ratfish. Like many carnivorous fishes, enzyme activities demonstrate that amino acids are metabolically important, although the concentration of plasma FAA was relatively low. NEFA concentrations are lower than in teleosts, but higher than in most elasmobranchs and similar to that in some "primitive" ray-finned fishes. NEFA composition is comparable to other marine temperate fishes, including high levels of n-6 and especially n-3 polyunsaturated fatty acids. The metabolic organization of the spotted ratfish is similar to that of elasmobranchs: a reduced capacity for lipid oxidation in muscle, lower plasma NEFA levels, and an emphasis on ketone bodies as oxidative fuel. This metabolic strategy was likely present in the common chondrichthyan ancestor, and may be similar to the ancestral metabolic state of fishes.     

Structural organization of the mitochondrial respiratory chain

Two models exist of the mitochondrial respiratory chain: the model of a random organization of the individual respiratory enzyme complexes and that of a super-complex assembly formed by stable association between the individual complexes. Recently Sch?gger, using digitonin solubilization and Blue Native PAGE produced new evidence of preferential associations, in particular a Complex I monomer with a Complex III dimer, and suggested a model of the respiratory chain (the respirasome) based on direct electron channelling between complexes. Discrimination between the two models is amenable to kinetic testing using flux control analysis. Experimental evidence obtained in beef heart SMP, according to the extension of the Metabolic Control Theory for pathways with metabolic channelling, showed that enzyme associations involving Complex I and Complex III take place in the respiratory chain while Complex IV seems to be randomly distributed, with cytochrome c behaving as a mobile component. Flux control analysis at anyone of the respiratory complexes involved in aerobic succinate oxidation indicated that Complex II and III are not functionally associated in a stable supercomplex. A critical appraisal of the solid-state model of the mitochondrial respiratory chain requires its reconciliation with previous biophysical and kinetic evidence that CoQ behaves as a homogeneous diffusible pool between all reducing enzyme and all oxidizing enzymes: the hypothesis can be advanced that both models (CoQ pool and supercomplexes) are true, by postulating that supercomplexes physiologically exist in equilibrium with isolated complexes depending on metabolic conditions of the cell.     

Short-chain 3-hydroxyacyl-coenzyme A dehydrogenase associates with a protein super-complex integrating multiple metabolic pathways

Proteins involved in mitochondrial metabolic pathways engage in functionally relevant multi-enzyme complexes. We previously described an interaction between short-chain 3-hydroxyacyl-coenzyme A dehydrogenase (SCHAD) and glutamate dehydrogenase (GDH) explaining the clinical phenotype of hyperinsulinism in SCHAD-deficient patients and adding SCHAD to the list of mitochondrial proteins capable of forming functional, multi-pathway complexes. In this work, we provide evidence of SCHAD's involvement in additional interactions forming tissue-specific metabolic super complexes involving both membrane-associated and matrix-dwelling enzymes and spanning multiple metabolic pathways. As an example, in murine liver, we find SCHAD interaction with aspartate transaminase (AST) and GDH from amino acid metabolic pathways, carbamoyl phosphate synthase I (CPS-1) from ureagenesis, other fatty acid oxidation and ketogenesis enzymes and fructose-bisphosphate aldolase, an extra-mitochondrial enzyme of the glycolytic pathway. Most of the interactions appear to be independent of SCHAD's role in the penultimate step of fatty acid oxidation suggesting an organizational, structural or non-enzymatic role for the SCHAD protein.     

Enzyme polymorphisms influencing the metabolism of heterocyclic aromatic amines

Heterocyclic aromatic amines are dietary carcinogens possibly involved in human carcinogenesis, DNA-adduct formation being an obligatory step in this multistage process. Heterocyclic amine binding to DNA largely depends on the balance between metabolic activation and detoxification pathways and DNA repair efficiency. Several genes coding for metabolic enzymes are polymorphic, which affects gene expression and/or enzyme activity. This paper briefly reviews the effect of polymorphisms of activating/detoxifying enzymes on the metabolism of heterocyclic amines. Despite some epidemiological evidence of an association between genetic polymorphisms and susceptibility to cancer possibly resulting from dietary exposure to heterocyclic aromatic amines (HA), the genetic polymorphisms had only slight effects on biomarker levels, suggesting the existence of further unknown factors.     

Understanding specificity in metabolic pathways—Structural biology of human nucleotide metabolism

Interactions are the foundation of life at the molecular level. In the plethora of activities in the cell, the evolution of enzyme specificity requires the balancing of appropriate substrate affinity with a negative selection, in order to minimize interactions with other potential substrates in the cell. To understand the structural basis for enzyme specificity, the comparison of structural and biochemical data between enzymes within pathways using similar substrates and effectors is valuable.Nucleotide metabolism is one of the largest metabolic pathways in the human cell and is of outstanding therapeutic importance since it activates and catabolises nucleoside based anti-proliferative drugs and serves as a direct target for anti-proliferative drugs. In recent years the structural coverage of the enzymes involved in human nucleotide metabolism has been dramatically improved and is approaching completion. An important factor has been the contribution from the Structural Genomics Consortium (SGC) at Karolinska Institutet, which recently has solved 33 novel structures of enzymes and enzyme domains in human nucleotide metabolism pathways and homologs thereof. In this review we will discuss some of the principles for substrate specificity of enzymes in human nucleotide metabolism illustrated by a selected set of enzyme families where a detailed understanding of the structural determinants for specificity is now emerging.     

Linear relationships between mitochondrial forces and cytoplasmic flows argue for the organized energy-coupled nature of cellular metabolism

We have studied rates of formation of glucose, urea and lactate by isolated hepatocytes incubated with a variety of inhibitors of energy transduction. Linear relationships have been found between these metabolic rates and mitochondrial forces (membrane, redox and phosphorylation potentials). The findings are suggestive of extensive enzyme organization within these metabolic pathways.     

Mutation-Selection Balance and Metabolic Control Theory

The evolution of metabolic control is examined with models that unify approaches of classical quantitative genetics and metabolic control theory. The quantitative traits considered are the activities of enzymes embedded within metabolic pathways. In the models, polygenic mutation alters the enzyme activities (Vmax/Km) according to prescribed distributions, and the population evolves following classical haploid viability selection. Stabilizing selection operates on global properties of the metabolic pathway, including either flux or metabolite pool concentration. Analytical results and numerical simulations demonstrate several important properties of these characters, including skewed, non-Gaussian equilibrium distributions, and an expected positive correlation between activities of enzymes flanking a substrate pool undergoing stabilizing selection. The house-of-cards approximation proved to be accurate in predicting the equilibrium distribution of allelic effects for a biologically reasonable segment of the parameter space. Further experimental and theoretical work is needed before a clear assessment can be made whether the observed variance in enzyme activities is explicable by a mutation-selection balance, and this system provides an excellent opportunity for such a test.     

Improving the phenotype predictions of a yeast genome‐scale metabolic model by incorporating enzymatic constraints

Genome‐scale metabolic models (GEMs) are widely used to calculate metabolic phenotypes. They rely on defining a set of constraints, the most common of which is that the production of metabolites and/or growth are limited by the carbon source uptake rate. However, enzyme abundances and kinetics, which act as limitations on metabolic fluxes, are not taken into account. Here, we present GECKO, a method that enhances a GEM to account for enzymes as part of reactions, thereby ensuring that each metabolic flux does not exceed its maximum capacity, equal to the product of the enzyme's abundance and turnover number. We applied GECKO to a Saccharomyces cerevisiae GEM and demonstrated that the new model could correctly describe phenotypes that the previous model could not, particularly under high enzymatic pressure conditions, such as yeast growing on different carbon sources in excess, coping with stress, or overexpressing a specific pathway. GECKO also allows to directly integrate quantitative proteomics data; by doing so, we significantly reduced flux variability of the model, in over 60% of metabolic reactions. Additionally, the model gives insight into the distribution of enzyme usage between and within metabolic pathways. The developed method and model are expected to increase the use of model‐based design in metabolic engineering.