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In the present article, we describe the two standard high-throughput methods for identification of protein complexes: two-hybrid screens and TAP (tandem affinity purification) tagging. These methods have been used to characterize the interactome of Saccharomyces cerevisiae, showing that the majority of proteins are part of complexes, and that complexes typically consist of a core to which are bound ‘party’ and ‘dater’ proteins. Complexes typically are merely the sum of their parts. A particularly interesting type of complex is the metabolon, containing enzymes within the same metabolic pathway. There is reasonably good evidence that metabolons exist, but they have not been detected using high-thoughput assays, possibly because of their fragility.

The soluble enzyme carbonic anhydrase II (CAII) plays an important role in CO(2) influx and efflux by red blood cells (RBCs), a process initiated by changes in the extracellular [CO(2)] (CO(2)-initiated CO(2) transport). Evidence suggests that CAII may be part of a macromolecular complex at the inner surface of the RBC membrane. Some have suggested CAII specifically binds to a motif on the cytoplasmic C terminus (Ct) of the Cl-HCO(3) exchanger AE1 and some other members of the SLC4 family of HCO(3)(-) transporters, a transport metabolon. Moreover, others have suggested that this bound CAII enhances the transport of HCO(3)(-)-related species-HCO(3)(-), CO(3)(), or CO(3)() ion pairs-when the process is initiated by altering the activity of the transporter (HCO(3)(-)-initiated HCO(3)(-) transport). In this review, I assess the theoretical roles of CAs in the transport of CO(2) and HCO(3)(-)-related species, concluding that although the effect of bound CAII on CO(2)-initiated CO(2) transport is expected to be substantial, the effect of bound CAs on HCO(3)(-)-initiated HCO(3)(-) transport is expected to be modest at best. I also assess the experimental evidence for CAII binding to AE1 and other transporters, and the effects of this binding on HCO(3)(-)-initiated HCO(3)(-) transport. The early conclusion that CAII binds to the Ct of AE1 appears to be the result of unpredictable effects of GST in the GST fusion proteins used in the studies. The early conclusion that bound CAII speeds HCO(3)(-)-initiated HCO(3)(-) transport appears to be the result of CAII accelerating the pH changes used as a read-out of transport. Thus, it appears that CAII does not bind directly to AE1 or other SLC4 proteins, and that bound CAII does not substantially accelerate HCO(3)(-)-initiated HCO(3)(-) transport.

The catabolic pathway for branched-chain amino acids includes deamination followed by oxidative decarboxylation of the deaminated product branched-chain α-keto acids, catalyzed by the mitochondrial branched-chain aminotransferase (BCATm) and branched-chain α-keto acid dehydrogenase enzyme complex (BCKDC). We found that BCATm binds to the E1 decarboxylase of BCKDC, forming a metabolon that allows channeling of branched-chain α-keto acids from BCATm to E1. The protein complex also contains glutamate dehydrogenase (GDH1), 4-nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1, pyruvate carboxylase, and BCKDC kinase. GDH1 binds to the pyridoxamine 5′-phosphate (PMP) form of BCATm (PMP-BCATm) but not to the pyridoxal 5′-phosphate-BCATm and other metabolon proteins. Leucine activates GDH1, and oxidative deamination of glutamate is increased further by addition of PMP-BCATm. Isoleucine and valine are not allosteric activators of GDH1, but in the presence of 5′-phosphate-BCATm, they convert BCATm to PMP-BCATm, stimulating GDH1 activity. Sensitivity to ADP activation of GDH1 was unaffected by PMP-BCATm; however, addition of a 3 or higher molar ratio of PMP-BCATm to GDH1 protected GDH1 from GTP inhibition by 50%. Kinetic results suggest that GDH1 facilitates regeneration of the form of BCATm that binds to E1 decarboxylase of the BCKDC, promotes metabolon formation, branched-chain amino acid oxidation, and cycling of nitrogen through glutamate.

The mitochondrial amidoxime reducing component (mARC) enzymes are molybdenum (Mo) -dependent oxidoreductases that function as part of a three-enzyme metabolon with cytochrome b5 (CYB5) and cytochrome b5 reductase (CYB5R). Previous studies in our lab have demonstrated that nitrite reduction to nitric oxide by the mARC-1 metabolon is regulated by oxygen via superoxide. However, the effect of oxygen on mARC-2, the mARC paralog expressed in human vascular tissue, has not been reported. Therefore, this study was conducted to determine the effect of oxygen on nitrite reduction to nitric oxide by mARC-2 and determine if superoxide is involved. We used purified recombinant mARC-2, CYB5, and CYBR enzymes and kinetic nitric oxide measurements to measure the effect of anoxia, hypoxia, and normoxia on nitrite reduction. We found that the NADH-dependent CYB5/CYB5R/mARC-2 metabolon is more sensitive to oxygen than the mARC-1 metabolon. However, the role of superoxide is less pronounced. This is in contrast with previous studies on the mARC-1 metabolon. Our data suggests that mARC-2 oxygen inhibition proceeds via a superoxide-independent mechanism, possibly direct and irreversible oxidation of the Mo active site.

Metabolomics is a technology for identifying and quantifying numerous biochemicals across metabolic pathways. Using this approach, we explored changes in biochemical profiles of human alveolar epithelial carcinoma (A549) cells following in vitro exposure to mainstream whole smoke (WS) aerosol as well as to wet total particulate matter (WTPM) or gas/vapor phase (GVP), the two constituent phases of WS from 2R4F Kentucky reference cigarettes. A549 cells were exposed to WTPM or GVP (expressed as WTPM mass equivalent GVP volumes) at 0, 5, 25, or 50 microg/mL or to WS from zero, two, four, and six cigarettes for 1 or 24 h. Cell pellets were analyzed for perturbations in biochemical profiles, with named biochemicals measured, analyzed, and reported in a heat map format, along with biochemical and physiological interpretations (mSelect, Metabolon Inc.). Both WTPM and GVP exposures likely decreased glycolysis (based on decreased glycolytic intermediaries) and increased oxidative stress and cell damage. Alterations in the Krebs cycle and the urea cycle were unique to WTPM exposure, while induction of hexosamines and alterations in lipid metabolism were unique to GVP exposure. WS altered glutathione (GSH) levels, enhanced polyamine and pantothenate levels, likely increased beta-oxidation of fatty acids, and increased phospholipid degradation marked by an increase in phosphoethanolamine. GSH, glutamine, and pantothenate showed the most significant changes with cigarette smoke exposure in A549 cells based on principal component analysis. Many of the changed biochemicals were previously reported to be altered by cigarette exposure, but the global metabolomic approach offers the advantage of observing changes to hundreds of biochemicals in a single experiment and the possibility for new discoveries. The metabolomic approach may thus be used as a screening tool to evaluate conventional and novel tobacco products offering the potential to reduce risks of smoking.

The phosphoenolpyruvate carboxylase (PEPC) interactome of developing castor oil seed (COS; Ricinus communis) endosperm was assessed using coimmunopurification (co-IP) followed by proteomic analysis. Earlier studies suggested that immunologically unrelated 107-kD plant-type PEPCs (p107/PTPC) and 118-kD bacterial-type PEPCs (p118/BTPC) are subunits of an unusual 910-kD hetero-octameric class 2 PEPC complex of developing COS. The current results confirm that a tight physical interaction occurs between p118 and p107 because p118 quantitatively coimmunopurified with p107 following elution of COS extracts through an anti-p107-IgG immunoaffinity column. No PEPC activity or immunoreactive PEPC polypeptides were detected in the corresponding flow-through fractions. Although BTPCs lack the N-terminal phosphorylation motif characteristic of PTPCs, Pro-Q Diamond phosphoprotein staining, immunoblotting with phospho-serine (Ser)/threonine Akt substrate IgG, and phosphate-affinity PAGE established that coimmunopurified p118 was multiphosphorylated at unique Ser and/or threonine residues. Tandem mass spectrometric analysis of an endoproteinase Lys-C p118 peptide digest demonstrated that Ser-425 is subject to in vivo proline-directed phosphorylation. The co-IP of p118 with p107 did not appear to be influenced by their phosphorylation status. Because p118 phosphorylation was unchanged 48 h following elimination of photosynthate supply due to COS depodding, the signaling mechanisms responsible for photosynthate-dependent p107 phosphorylation differ from those controlling p118's in vivo phosphorylation. A 110-kD PTPC coimmunopurified with p118 and p107 when depodded COS was used. The plastidial pyruvate dehydrogenase complex (PDCpl) was identified as a novel PEPC interactor. Thus, a putative metabolon involving PEPC and PDCpl could function to channel carbon from phosphoenolpyruvate to acetyl-coenzyme A and/or to recycle CO2 from PDCpl to PEPC.

Plants produce a plethora of secondary metabolites which constitute a wealth of potential pharmaceuticals, pro-vitamins, flavours, fragrances, colorants and toxins as well as a source of natural pesticides. Many of these valuable compounds are only synthesized in exotic plant species or in concentrations too low to facilitate commercialization. In some cases their presence constitutes a health hazard and renders the crops unsuitable for consumption. Metabolic engineering is a powerful tool to alter and ameliorate the secondary metabolite composition of crop plants and gain new desired traits. The interplay of a multitude of biosynthetic pathways and the possibility of metabolic cross-talk combined with an incomplete understanding of the regulation of these pathways, explain why metabolic engineering of plant secondary metabolism is still in its infancy and subject to much trial and error. Cyanogenic glucosides are ancient defense compounds that release toxic HCN upon tissue disruption caused e.g. by chewing insects. The committed steps of the cyanogenic glucoside biosynthetic pathway are encoded by three genes. This unique genetic simplicity and the availability of the corresponding cDNAs have given cyanogenic glucosides pioneering status in metabolic engineering of plant secondary metabolism. In this review, lessons learned from metabolic engineering of cyanogenic glucosides in Arabidopsis thaliana (thale cress), Nicotiana tabacum cv Xanthi (tobacco), Manihot esculenta Crantz (cassava) and Lotus japonicus (bird's foot trefoil) are presented. The importance of metabolic channelling of toxic intermediates as mediated by metabolon formation in avoiding unintended metabolic cross-talk and unwanted pleiotropic effects is emphasized. Likewise, the potential of metabolic engineering of plant secondary metabolism as a tool to elucidate, for example, the impact of secondary metabolites on plant-insect interactions is demonstrated

Association of some plasma membrane bicarbonate transporters with carbonic anhydrase enzymes forms a bicarbonate transport metabolon to facilitate metabolic CO2-HCO3−conversions and coupled HCO3−transport. The transmembrane carbonic anhydrase, CAIX, with its extracellular catalytic site, is highly expressed in parietal and other cells of gastric mucosa, suggesting a role in acid secretion. We examined in transfected HEK293 cells the functional and physical interactions between CAIX and the parietal cell Cl−/HCO3−exchanger AE2 or the putative Cl−/HCO3−exchanger SLC26A7. Coexpression of CAIX increased AE2 transport activity by 28 ± 7% and also activated transport mediated by AE1 and AE3 (32 ± 10 and 37 ± 9%, respectively). In contrast, despite a transport rate comparable to that of AE3, coexpressed CAIX did not alter transport associated with SLC26A7. The CAIX-associated increase of AE2 activity did not result from altered AE2 expression or cell surface processing. CAIX was coimmunoprecipitated with the coexpressed SLC4 polypeptides AE1, AE2, and AE3, but not with SLC26A7. GST pull-down assays with a series of domain-deleted forms of CAIX revealed that the catalytic domain of CAIX mediated interaction with AE2. AE2 and CAIX colocalized in human gastric mucosa, as indicated by coimmunofluorescence. This is the first example of a functional and physical interaction between a bicarbonate transporter and a transmembrane carbonic anhydrase. We conclude that CAIX can bind to some Cl−/HCO3−exchangers to form a bicarbonate transport metabolon.

We have demonstrated the accuracy of a spatial stochastic model of Escherichia coli central carbon metabolism using the next subvolume method (NSM), an efficient implementation of the Gillespie direct method of stochastic simulation. Using this model, we demonstrate that compartmentalization of the enzymes comprising an engineered pathway for biosynthesis of R-1,2-propanediol leads to improved kinetic properties for the pathway enzymes, especially when substrate diffusivities are low. Our results suggest that enzyme compartmentalization is a powerful approach for improving the catalytic turnover of a channeled carbon substrate and should be particularly useful when applied to synthetic metabolic pathways that suffer from poor translation efficiency, are present in highly variable copy numbers, and have low turnover for new substrates. Furthermore, this approach represents a generic modeling framework for simultaneously analyzing spatial and stochastic events in cellular metabolism and should enable quantitative evaluation of the effect of enzyme compartmentalization on virtually any recombinant pathway.

Several acid/base-coupled membrane transporters, such as the electrogenic sodium-bicarbonate cotransporter (NBCe1), have been shown to bind to different carbonic anhydrase isoforms to create a “transport metabolon.” We have expressed NBCe1 derived from human kidney in oocytes of Xenopus leavis and determined its transport activity by recording the membrane current in voltage clamp, and the cytosolic H+ and Na+ concentrations using ion-selective microelectrodes. When carbonic anhydrase isoform II (CAII) had been injected into oocytes, the membrane current and the rate of cytosolic Na+ rise, indicative for NBCe1 activity, increased significantly with the amount of injected CAII (2–200 ng). The CAII inhibitor ethoxyzolamide reversed the effects of CAII on the NBCe1 activity. Co-expressing wild-type CAII or NH2-terminal mutant CAII together with NBCe1 provided similar results, whereas co-expressing the catalytically inactive CAII mutant V143Y had no effect on NBCe1 activity. Mass spectrometric analysis and the rate of cytosolic H+ change following addition of CO2/ confirmed the catalytic activity of injected and expressed CAII in oocytes. Our results show that the transport capacity of NBCe1 is enhanced by the catalytic activity of CAII, in line with the notion that CAII forms a transport metabolon with NBCe1.

The catabolic pathways of branched-chain amino acids have two common steps. The first step is deamination catalyzed by the vitamin B(6)-dependent branched-chain aminotransferase isozymes (BCATs) to produce branched-chain alpha-keto acids (BCKAs). The second step is oxidative decarboxylation of the BCKAs mediated by the branched-chain alpha-keto acid dehydrogenase enzyme complex (BCKD complex). The BCKD complex is organized around a cubic core consisting of 24 lipoate-bearing dihydrolipoyl transacylase (E2) subunits, associated with the branched-chain alpha-keto acid decarboxylase/dehydrogenase (E1), dihydrolipoamide dehydrogenase (E3), BCKD kinase, and BCKD phosphatase. In this study, we provide evidence that human mitochondrial BCAT (hBCATm) associates with the E1 decarboxylase component of the rat or human BCKD complex with a K(D) of 2.8 microM. NADH dissociates the complex. The E2 and E3 components do not interact with hBCATm. In the presence of hBCATm, k(cat) values for E1-catalyzed decarboxylation of the BCKAs are enhanced 12-fold. Mutations of hBCATm proteins in the catalytically important CXXC center or E1 proteins in the phosphorylation loop residues prevent complex formation, indicating that these regions are important for the interaction between hBCATm and E1. Our results provide evidence for substrate channeling between hBCATm and BCKD complex and formation of a metabolic unit (termed branched-chain amino acid metabolon) that can be influenced by the redox state in mitochondria.

Based on solid-phase binding assays with enzyme-linked immunosorbent assay detection, previous investigators suggested that intracellular carbonic anhydrase II (CA II) interacts at high affinity with the C-terminal (Ct) domains of SLC4 bicarbonate-transport proteins, expressed as glutathione S-transferase (GST) fusion proteins, to form functional HCO3- metabolons. Here we re-evaluated this protein-protein interaction using two solid-phase binding assays. We first compared the ability of the Ct domain of three SLC4 transporters, SLC4-A1 (AE1), SLC4-A4 (NBCe1), and SLC4-A8 (NDCBE), to bind immobilized CA II, using enzyme-linked immunosorbent assay detection. We found that when expressed as GST fusion proteins, all three bind to CA II (Kd 300-600 nM) better than does pure GST. However, we detected no binding of pure SLC4-Ct peptides to immobilized CA II. Second, we reversed assay orientation by immobilizing the SLC4-Ct fusion proteins or peptides. We found that more CA II binds to GST than to any of the three GST-SLC4-Ct fusion proteins. Furthermore, we detected no binding of CA II to any of the immobilized pure SLC4-Ct peptides. Finally, we used surface plasmon resonance to detect possible rapid interactions between CA II and the pure peptides. Although we detected acetazolamide binding to immobilized CA II and specific antibodies binding to immobilized SLC4-Ct peptides, we detected no binding of CA II to immobilized SLC4-Ct or vice versa. Thus, although an HCO3 metabolon may exist, CA II cannot bind directly to pure SLC4-Ct peptides and can bind to GST-SLC4-Ct fusion proteins only when the CA II is immobilized and the fusion protein is soluble, and not vice versa.

Carbonic anhydrase (CA) catalyzes the reversible hydration of CO(2). CA is expressed in most segments of the kidney. CAII and CAIV predominate in human and rabbit kidneys; in rodent kidneys, CAXII, and CAXIV are also present. CAIX is expressed by renal cell carcinoma (RCC). Most of these isoforms, except for rodent CAIV, have high turnover rates. CAII is a cytoplasmic enzyme, whereas the others are membrane-associated; CAIV is anchored by glycosylphosphatidylinositol linkage. Membrane polarity is apical for CAXIV, basolateral for CAXII, and apical and basolateral for CAIV. Luminal membrane CAs facilitate the dehydration of carbonic acid (H(2)CO(3)) that is formed when secreted protons combine with filtered bicarbonate. Basolateral CA enhances the efflux of bicarbonate via dehydration of H(2)CO(3). CAII and CAIV can associate with bicarbonate transporters (e.g., AE1, kNBC1, NBC3, and SCL26A6), and proton antiporter, NHE1 in a membrane protein complex called a transport metabolon. CAXII and CAXIV may also be associated with transporters in normal kidney and CAIX in RCCs. The multiplicity of CAs implicates their importance in acid-base and other solute transport along the nephron. For example, CAII on the cytoplasmic face and CAIV on the extracellular surface provide the 'push' and 'pull' for bicarbonate transport by supplying and dissipating substrate respectively.

Cellular NAD(P)H-dependent oxidoreductase activity with artificial dyes (NAD(P)H-OR) is an indicator of viability, as the cellular redox state is important for biosynthesis and antioxidant defense. However, high NAD(P)H due to impaired mitochondrial oxidation, known as reductive stress, should increase NAD(P)H-OR yet perturb viability. To better understand this complex behavior, we assayed NAD(P)H-OR with resazurin (Alamar Blue) in glioblastoma cell lines U87 and T98G, treated with inhibitors of central metabolism, oxythiamin, and phosphonate analogs of 2-oxo acids. Targeting the thiamin diphosphate (ThDP)-dependent enzymes, the inhibitors are known to decrease the NAD(P)H production in the pentose phosphate shuttle and/or upon mitochondrial oxidation of 2-oxo acids. Nevertheless, the inhibitors elevated NAD(P)H-OR with resazurin in a time- and concentration-dependent manner, suggesting impaired NAD(P)H oxidation rather than increased viability. In particular, inhibition of the ThDP-dependent enzymes affects metabolism of malate, which mediates mitochondrial oxidation of cytosolic NAD(P)H. We showed that oxythiamin not only inhibited mitochondrial 2-oxo acid dehydrogenases, but also induced cell-specific changes in glutamate and malate dehydrogenases and/or malic enzyme. As a result, inhibition of the 2-oxo acid dehydrogenases compromises mitochondrial metabolism, with the dysregulated electron fluxes leading to increases in cellular NAD(P)H-OR. Perturbed mitochondrial oxidation of NAD(P)H may thus complicate the NAD(P)H-based viability assay.

Anodic bioelectrodes for biofuel cells are more complex than cathodic bioelectrodes for biofuel cells, because laccase and bilirubin oxidase can individually catalyze four electron reduction of oxygen to water, whereas most anodic enzymes only do a single two electron oxidation of a complex fuel (i.e. glucose oxidase oxidizing glucose to gluconolactone while generating 2 electrons of the total 24 electrons), so enzyme cascades are typically needed for complete oxidation of the fuel. This review article will discuss the lessons learned from natural metabolic pathways about multi-step oxidation and how those lessons have been applied to minimal or artificial enzyme cascades. This article is part of a Special Issue entitled Biodesign for Bioenergetics--the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson.

Cholesterol is essential to human health, and its levels are tightly regulated by a balance of synthesis, uptake, and efflux. Cholesterol synthesis requires the actions of more than twenty enzymes to reach the final product, through two alternate pathways. Here we describe a physical and functional interaction between the two terminal enzymes. 24-Dehydrocholesterol reductase (DHCR24) and 7-dehydrocholesterol reductase (DHCR7) coimmunoprecipitate, and when the DHCR24 gene is knocked down by siRNA, DHCR7 activity is also ablated. Conversely, overexpression of DHCR24 enhances DHCR7 activity, but only when a functional form of DHCR24 is used. DHCR7 is important for both cholesterol and vitamin D synthesis, and we have identified a novel layer of regulation, whereby its activity is controlled by DHCR24. This suggests the existence of a cholesterol #x201C;metabolon#x201D;, where enzymes from the same metabolic pathway interact with each other to provide a substrate channeling benefit. We predict that other enzymes in cholesterol synthesis may similarly interact, and this should be explored in future studies.

In diseases where glucose utilization is impaired ketone bodies and TCA cycle intermediates serve as alternative fuel. Since these diseases are linked pathophysiologically to impaired mitochondrial function, we hypothesized that nutritional ketosis induced by ketogenic precursors or TCA cycle intermediate supplements promote mitochondrial biogenesis and preservation of existing mitochondria. Exogenous ketone supplementation was used in Sprague Dawley (SD) rats and in SOD1 G93A mice. In SD rats, ketone ester and ketone salt-MCT oil mixture was gavaged daily, whereas the Deanna protocol (DP) supplementation containing tricarboxylic acid (TCA) cycle intermediates was added to standard rodent chow fed to SOD1 G93A mice. Plasma was collected at endpoint and global metabolomic profiles were compared by Metabolon. Morphology, density and distribution of skeletal muscle mitochondria were studied by TEM and SEM. Exogenous ketone supplementation and the DP supplementation dose-dependently increased compounds associ...

Isoflavonoids are specialized metabolites, almost exclusive to the legume family of plants. They are actors in symbiosis with nitrogen-fixing bacteria and in plant stress response. Isoflavonoids are noted for their human health benefits. Isoflavonoid content in legumes has proven to be a complex trait. The goal of the present research is to determine the mechanisms underlying isoflavonoid biosynthesis in soybean. The first approach was to unravel the genetic factors of isoflavonoid biosynthesis. A branch-point enzyme of the phenylpropanoid pathway, chalcone isomerase (CHI), catalyzes the reaction producing flavanones, the nucleus for many downstream metabolites such as isoflavonoids. I identified twelve soybean CHI genes, including five new members. The evolutionary history of the family shows that the enzymatic fold evolved from being catalytically inactive to being a chalcone-to-flavanone isomerase. Four CHIs in soybean were identified with the latter functionality. The CHIs showed differential temporal and spatial expression, pointing to the potential function of CHI1A in soybean seed isoflavonoid production. On the molecular level of organization, the long-postulated model of a subcellular isoflavonoid enzyme complex forming at the surface of the endoplasmic reticulum (ER) was substantiated in the present study. Here, I identified key players in the ‘metabolon’, established in planta subcellular localization, and investigated protein-protein interaction. The results suggest that isoflavone synthase (IFS), a cytochrome P450, is a nucleating metabolic center at the surface of the ER, interacting with upstream pathway enzymes. Finally, a transcriptomic study was undertaken to find genetic elements linked with isoflavonoid content variation in four soybean cultivars. The results suggest that competing branches of the phenylpropanoid pathway are combinatorially regulated to coordinate flux into isoflavonoid biosynthesis. The candidate genes encode enzymes in the overlapping pathways, several transcription factors, metabolic transporters and more. Study of the CHI gene family and isoflavonoid biosynthesis has provided us with new insights into production and regulation of this important plant natural product. This knowledge can facilitate the manipulation of metabolic content and composition in legumes, and introduction of the isoflavonoid pathway into non-legume crops.

Bitter acids (α and β types) account for more than 30% of the fresh weight of hop (Humulus lupulus) glandular trichomes and are well known for their contribution to the bitter taste of beer. These multiprenylated chemicals also show diverse biological activities, some of which have potential benefits to human health. The bitter acid biosynthetic pathway has been investigated extensively, and the genes for the early steps of bitter acid synthesis have been cloned and functionally characterized. However, little is known about the enzyme(s) that catalyze three sequential prenylation steps in the β-bitter acid pathway. Here, we employed a yeast (Saccharomyces cerevisiae) system for the functional identification of aromatic prenyltransferase (PT) genes. Two PT genes (HlPT1L and HlPT2) obtained from a hop trichome-specific complementary DNA library were functionally characterized using this yeast system. Coexpression of codon-optimized PT1L and PT2 in yeast, together with upstream genes, led to the production of bitter acids, but no bitter acids were detected when either of the PT genes was expressed by itself. Stepwise mutation of the aspartate-rich motifs in PT1L and PT2 further revealed the prenylation sequence of these two enzymes in β-bitter acid biosynthesis: PT1L catalyzed only the first prenylation step, and PT2 catalyzed the two subsequent prenylation steps. A metabolon formed through interactions between PT1L and PT2 was demonstrated using a yeast two-hybrid system, reciprocal coimmunoprecipitation, and in vitro biochemical assays. These results provide direct evidence of the involvement of a functional metabolon of membrane-bound prenyltransferases in bitter acid biosynthesis in hop.

The three branched chain amino acids are unique in that the first catabolic step does not occur in the liver. Leucine, isoleucine, and valine share the first two catabolic enzymes—branched chain aminotransferase (BCAT) and branched chain ketoacid dehydrogenase (BCKD). Both BCAT isoforms use Vitamin B-6 cofactors as temporary acceptors of the α-amino group during the process of aminating α-ketoglutarate, which becomes glutamate. The deaminated BCAAs are known as BCKAs, and can be reaminated into their BCAA form. The next step in BCAA catabolism, the decarboxylation of the BCKAs, is irreversible. In some tissues, the mitochondrial isoform of BCAT (BCATm), appears to form a metabolon with BCKD, which is a physical relationship between the BCATm enzyme and the BCKD enzyme complex. This facilitates the transfer of substrates and increases the efficiency of the reactions. After decarboxylation of the BCKA, the catabolic pathways for the three BCAA precursors diverge, utilizing different enzymes and processes. Disruptions in BCAA metabolism, primarily occurring in the first two steps, can cause a number of severe metabolic disorders. Additionally, changes in both BCAAs and BCAA enzymes have been implicated in the pathophysiology of TBI.

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.

Isoprenoids are the most diverse and abundant group of natural products. In plants, farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) are precursors to many isoprenoids having essential functions. Terpenoids and sterols are derived from FPP, whereas gibberellins, carotenoids, casbenes, taxenes, and others originate from GGPP. The corresponding synthases (FPP synthase [FPPS] and GGPP synthase [GGPPS]) catalyze, respectively, the addition of two and three isopentenyl diphosphate molecules to dimethylallyl diphosphate. Maize (Zea mays L. cv B73) endosperm cDNAs encoding isoprenoid synthases were isolated by functional complementation of Escherichia coli cells carrying a bacterial gene cluster encoding all pathway enzymes needed for carotenoid biosynthesis, except for GGPPS. This approach indicated that the maize gene products were functional GGPPS enzymes. Yet, the predicted enzyme sequences revealed FPPS motifs and homology with FPPS enzymes. In vitro assays demonstrated that indeed these maize enzymes produced both FPP and GGPP and that the N-terminal sequence affected the ratio of FPP to GGPP. Their functionality in E. coli demonstrated that these maize enzymes can be coupled with a metabolon to provide isoprenoid substrates for pathway use, and suggests that enzyme bifunctionality can be harnessed. The maize cDNAs are encoded by a small gene family whose transcripts are prevalent in endosperm beginning mid development. These maize cDNAs will be valuable tools for assessing the critical structural properties determining prenyl transferase specificity and in metabolic engineering of isoprenoid pathways, especially in cereal crops.

The exceptionally high protein concentration in living cells can favor functional protein-protein interactions that can be difficult to detect with purified proteins. In this study we describe specific interactions between mammalian D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and L-lactate dehydrogenase (LDH) isozymes from heart and muscle. We use poly(ethylene-glycol) (PEG)-induced coprecipitation and native agarose electrophoresis as two independent methods uniquely suited to mimic some of the conditions that can favor protein-protein interaction in living cells. We found that GAPDH interacts with heart or muscle isozymes of LDH with approximately one-to-one stoichiometry. The interaction is specific; GAPDH shows interaction with two LDH isozymes that have very different net charge and solubility in PEG solution, while no interaction is observed with GAPDH from other species, other NAD(H) dehydrogenases, or other proteins that have very similar net charge and molecular mass. Analytical ultracentrifugation showed that the LDH and GAPDH complex is insoluble in PEG solution. The interaction is abolished by saturation with NADH, but not by saturation with NAD(+) in correlation with GAPDH solubility in PEG solution. The crystal structures show that GAPDH and LDH isozymes share complementary size, shape, and electric potential surrounding the active sites. The presented results suggest that GAPDH and LDH have a functional interaction that can affect NAD(+)/NADH metabolism and glycolysis in living cells.

Dynamics of metabolon formation in mitochondria was probed by studying diffusional motion of two sequential Krebs cycle enzymes in a microfluidic channel. Enhanced directional co-diffusion of both enzymes against a substrate concentration gradient was observed in the presence of intermediate generation. This reveals a metabolite directed compartmentation of metabolic pathways.

Flavonoids, some natural compounds exhibiting antioxidant properties, are synthesized along enzymatic cascades involving several enzymes. It has been recently proposed that some of these enzymes are involved in the formation of large transient macromolecular edifices, called metabolon, interacting with cellular membrane. Such molecular complexes should allow direct metabolites transfer from one active site to the other (substrate channeling phenomenon), minimizing diffusion time and solvation effects. The present work aims to establish a first model of a metabolon involving 3 enzymes of the flavonoid biosynthesis: the dihydroflavonol 4-reductase (DFR), the flavonoid 3'-hydroxylase (F3'H) and the leucoanthocyanidin reductase (LAR). The study of such large molecular system requires a multiscale approach based on i) hybrid QM/MM methods to decipher enzymatic mechanism, ii) molecular dynamic simulations in microsecond timescale to estimate thermodynamic and kinetic properties and iii) protein-protein docking at coarse-grained resolution. These different levels of theory allow us to establish a first model of a three-enzymes-metabolon in interaction with a model of cellular membrane., Les flavonoïdes, molécules naturelles possédant des propriétés antiradicalaires et antioxydantes, sont produits au cours de cascades enzymatiques impliquant plusieurs enzymes. Il a récemment été proposé que certaines de ces enzymes s'assembleraient pour former un complexe multienzymatique transitoire, appelé métabolon, ancré à la membrane cellulaire. Cette structure rendrait possible des phénomènes de transfert direct de métabolites d'un site actif à l'autre (substrate channeling), réduisant ainsi les temps de diffusion et minimisant les effets de solvatation/désolvatation du substrat. L'objectif de ce travail est de proposer un premier modèle de ce type de complexe, impliquant trois enzymes de la biosynthèse des flavonoïdes : la dihydroflavonol-4-réductase (DFR), la flavonoïd-3'-hydroxylase (F3’H) et la leucoanthocyanidine réductase (LAR). L'étude de ces complexes moléculaires requiert la mise en œuvre d'une méthodologie multi-échelles basée sur l’utilisation i) de méthodes hybrides QM/MM pour l'étude de la réactivité enzymatique, ii) de simulations de dynamique moléculaire à résolution atomique se déroulant sur des échelles de temps de l'ordre de la microseconde pour estimer des propriétés thermodynamiques et cinétiques, iii) de calculs de docking protéine-protéine en résolution gros grain. L'application des différents niveaux de théorie nous a permis d'établir un premier modèle de métabolon à trois enzymes en interaction avec une membrane cellulaire.

The euryhaline pupfish, Cyprinodon variegatus variegatus ( Cvv ), can successfully osmoregulate in ≥ 2mM Na + and a freshwater population ( Cyprinodon variegatus hubbsi; Cvh ) osmoregulates at ≥ 0.1mM Na + . We previously demonstrated that Cvv relies on an apical NKCC and NHE in the gill for Na + uptake in high (7mM) and intermediate (2mM) Na + concentrations, while Cvh relies only on NHE for Na + uptake. This study investigated whether differential NHE isoform use explains differences in Na + uptake kinetics between these two populations. We further studied whether Cvh uses a NHE-Rh metabolon or carbonic anhydrase (CA) to overcome thermodynamic challenges of NHE function in dilute freshwater. Transfer to more dilute freshwater resulted in upregulation of nhe-2 ( Cvv only) and nhe-3 ( Cvv and Cvh ). Relative expression of nhe-3 compared to nhe-2 was 2-fold higher in Cvv , but 200-fold higher in Cvh suggesting that nhe-3 expression is an important freshwater adaptation for Cvh . Simultaneous measurement of Na + and T amm flux under various conditions provided no support for a NHE-Rh metabolon in either population. Carbonic anhydrase activity in Cvv was comparable in 7 and 2mM Na + acclimated fish. In Cvh , CA activity increased by 75% in 0.1mM Na + acclimated fish compared to 7mM Na + fish. Ethoxzolamide had variable effects, stimulating and reducing Na + uptake in Cvv acclimated to 7 and 2mM Na + , while reducing Na + uptake in 7 and 0.1mM Na + acclimated Cvh . This suggests that CA plays important, but different roles in regulating Na + uptake in Cvv and Cvh .

Carbonic anhydrases (CA) catalyze the reversible conversion of CO2 to HCO3-. Some bicarbonate transporters bind CA, forming a complex called a transport metabolon, to maximize the coupled catalytic/transport flux. SLC26A6, a plasma membrane Cl-/HCO3- exchanger with a suggested role in pancreatic HCO3- secretion, was found to bind the cytoplasmic enzyme CAII. Mutation of the identified CAII binding (CAB) site greatly reduced SLC26A6 activity, demonstrating the importance of the interaction. Regulation of SLC26A6 bicarbonate transport by protein kinase C (PKC) was investigated. Angiotensin II (AngII), which activates PKC, decreased Cl-/HCO3- exchange in cells coexpressing SLC26A6 and AT1a-AngII receptor. Activation of PKC reduced SLC26A6/CAII association in immunoprecipitates. Similarly, PKC activation displaced CAII from the plasma membrane, as monitored by immunofluorescence. Finally, mutation of a PKC site adjacent to the SLC26A6 CAB site rendered the transporter unresponsive to PKC. PKC therefore reduces CAII/SLC26A6 interaction, reducing bicarbonate transport rate. Taken together, our data support a mechanism for acute regulation of membrane transport: metabolon disruption.

A pathway intermediate is said to be ‘channeled’ when an intermediate just made in a pathway has a higher probability of being a substrate for the next pathway enzyme compared with a molecule of the same species from the aqueous cytoplasm. Channeling is an important phenomenon because it might play a significant role in the regulation of metabolism. Whereas the usual mechanism proposed for channeling is the (often) transient interaction of sequential pathway enzymes, many of the supporting data come from results with pure enzymes and dilute cell extracts. Even when isotope dilution techniques have utilized whole-cell systems, most often only a qualitative assessment of channeling has been reported. Here we develop a method for making a quantitative calculation of the fraction channeled in glycolysis from in vivo isotope dilution experiments. We show that fructose-1,6-bisphosphate, in whole cells of Escherichia coli, was strongly channeled all the way to CO2, whereas fructose-6-phosphate was not. Because the signature of channeling is lost if any downstream intermediate prior to CO2 equilibrates with molecules in the aqueous cytosol, it was not possible to evaluate whether glucose-6-phosphate was channeled in its transformation to fructose-6-phosphate. The data also suggest that, in addition to pathway enzymes being associated with one another, some are free in the aqueous cytosol. How sensitive the degree of channeling is to growth or experimental conditions remains to be determined.

To characterize the location of glycolytic enzymes (GEs) in intact human erythrocytes, freshly drawn blood was fixed and stained with Abs to GAPDH, aldolase, phosphofructokinase (PFK), pyruvate kinase (PK), lactate dehydrogenase (LDH), carbonic anhydrase II, Hb, and band 3 (AE1). Confocal microscopy revealed that in cells where band 3 displays its expected membrane staining and Hb is evenly distributed across the cytoplasm, GEs are largely limited to the membrane. Biochemical studies confirmed that the membrane binding sites for GAPDH, aldolase, and PFK reside on band 3, but related analyses demonstrate that sites for PK and LDH do not. Four lines of evidence demonstrate that the GEs are at least partially assembled into multimeric complexes near the NH 2 terminus of band 3. First, a mAb to residues 1–12 of band 3 displaces all of the above GEs from the membrane, including LDH and PK, which do not bind band 3. Second, tyrosine phosphorylation of the NH 2 terminus of band 3 (Y8 and Y21) reversibly releases all of the GEs from the membrane, including LDH and PK. Third, deoxygenation of RBCs dislodges all GEs from the membrane, consistent with the established ability of deoxyHb but not oxyHb to bind the NH 2 terminus of band 3. Fourth, a large increase in the accessibility of enzyme epitopes is observed upon dissociation of GEs from the membrane. We conclude, therefore, that GEs are organized into complexes on the membrane whose assembly is regulated by oxygenation and phosphorylation.

The biosynthesis of structural and signaling molecules depends on intracellular concentrations of essential amino acids, which are maintained by a specific system of plasma membrane transporters. We identify a unique population of nutrient amino acid transporters (NATs) within the sodium-neurotransmitter symporter family and have characterized a member of the NAT subfamily from the larval midgut of the Yellow Fever vector mosquito, Aedes aegypti (aeAAT1, AAR08269), which primarily supplies phenylalanine, an essential substrate for the synthesis of neuronal and cuticular catecholamines. Further analysis suggests that NATs constitute a comprehensive transport metabolon for the epithelial uptake and redistribution of essential amino acids including precursors of several neurotransmitters. In contrast to the highly conserved subfamily of orthologous neurotransmitter transporters, lineage-specific, paralogous NATs undergo rapid gene multiplication/substitution that enables a high degree of evolutionary plasticity of nutrient amino acid uptake mechanisms and facilitates environmental and nutrient adaptations of organisms. These findings provide a unique model for understanding the molecular mechanisms, physiology, and evolution of amino acid and neurotransmitter transport systems and imply that monoamine and GABA transporters evolved by selection and conservation of earlier neuronal NATs.

Thiamine (Vitamin B1) deficiency (TD) leads to memory deficits and neurological disease in animals and humans. The thiamine-dependent enzymes of the tricarboxylic acid (TCA) cycle are reduced following TD and in the brains of patients that died from multiple neurodegenerative diseases. Whether reductions in thiamine or thiamine-dependent enzymes leads to changes in all TCA cycle enzymes has never been tested. In the current studies, the pyruvate dehydrogenase complex (PDHC) and all of enzymes of the TCA cycle were measured in the brains of TD mice. Non-thiamine-dependent enzymes such as succinate dehydrogenase (SDH), succinate thiokinase (STH) and malate dehydrogenase (MDH) were altered as much or more than thiamine-dependent enzymes such as the alpha-ketoglutarate dehydrogenase complex (KGDHC) (-21.5%) and PDHC (-10.5%). Succinate dehydrogenase (SDH) activity decreased by 27% and succinate thiokinase (STH) decreased by 24%. The reductions in these other enzymes may result from oxidative stress because of TD or because these other enzymes of the TCA cycle are part of a metabolon that respond as a group of enzymes. The results suggest that other TCA cycle enzymes should be measured in brains from patients that died from neurological disease in which thiamine-dependent enzymes are known to be reduced. The diminished activities of multiple TCA cycle enzymes may be important in our understanding of how metabolic lesions alter brain function in neurodegenerative disorders.

The fifth step of histidine biosynthesis is catalysed by an imidazole glycerol-phosphate (IGP) synthase. In Archaea and Bacteria, the active form of IGP synthase is a stable 1:1 dimeric complex constituted by a glutamine amidotransferase (GAT) and a cyclase, the products of hisH and hisF. In Eucarya, the two activities are associated with a single bifunctional polypeptide encoded by HIS7. In this work, we report a comparative analysis of the amino acid sequence of all the available HisH, HisF and HIS7 proteins, which allowed depicting a likely evolutionary pathway leading to the present-day bifunctional HIS7 genes. According to the model that we propose, the bifunctional HIS7 gene is the outcome of a gene fusion event between two independent ancestral cistrons encoding an amidotransferase and a cyclase, respectively. The phylogenetic distribution of the eucaryal HIS7 genes and the analysis of all the available prokaryotic counterparts (hisH and hisF) revealed the absence of such fusions in prokaryotes, suggesting that the fusion event very likely occurred in an early stage of eucaryal evolution and was fixed in the nucleated cells. The biological significance of this gene fusion is also discussed.

We have recently shown that carbonic anhydrase II (CAII) binds in vitro to the C-terminus of the electrogenic sodium bicarbonate cotransporter kNBC1 (kNBC1-ct). In the present study we determined the molecular mechanisms for the interaction between the two proteins and whether kNBC1 and CAII form a transport metabolon in vivo wherein bicarbonate is transferred from CAII directly to the cotransporter. Various residues in the C-terminus of kNBC1 were mutated and the effect of these mutations on both the magnitude of CAII binding and the function of kNBC1 expressed in mPCT cells was determined. Two clusters of acidic amino acids, L958DDV and D986NDD in the wild-type kNBC1-ct involved in CAII binding were identified. In both acidic clusters, the first aspartate residue played a more important role in CAII binding than others. A significant correlation between the magnitude of CAII binding and kNBC1-mediated flux was shown. The results indicated that CAII activity enhances flux through the cotransporter when the enzyme is bound to kNBC1. These data are the first direct evidence that a complex of an electrogenic sodium bicarbonate cotransporter with CAII functions as a transport metabolon.

The terminal steps involved in making ATP in mitochondria require an ATP synthase (F(0)F(1)) comprised of two motors, a phosphate carrier (PIC), and an adenine nucleotide carrier (ANC). Under mild conditions, these entities sub-fractionate as an ATP synthase/PIC/ANC complex or "ATP synthasome" (Ko, Y.H., Delannoy, M, Hullihen, J., Chiu, W., and Pedersen, P.L. (2003) J. Biol. Chem. 278, 12305-12309). As a first step toward obtaining three-dimensional information about this large complex or "metabolon" and the locations of PIC and ANC therein, we dispersed ATP synthasomes into single complexes and visualized negatively stained images by electron microscopy (EM) that showed clearly the classical headpiece, central stalk, and basepiece. Parallel immuno-EM studies revealed the presence of PIC and ANC located non-centrally in the basepiece, and other studies implicated an ATP synthase/PIC/ANC stoichiometry near 1:1:1. Single ATP synthasome images (7506) were boxed, and, using EMAN software, a three-dimensional model was obtained at a resolution of 23 A. Significantly, the basepiece is oblong and contains two domains, the larger of which connects to the central stalk, whereas the smaller appears as an extension. Docking studies with known structures together with the immuno-EM studies suggest that PIC or ANC may be located in the smaller domain, whereas the other transporter resides nearby in the larger domain. Collectively, these finding support a mechanism in which the entry of the substrates ADP and P(i) into mitochondria, the synthesis of ATP on F(1), and the release and exit of ATP are very localized and highly coordinated events.

A series of positively charged sulfonamides were obtained by reaction of aminobenzolamide [5-(4-aminobenzenesulfonylamino)-1,3,4-thiadiazole-2-sulfonamide] with tri-/tetrasubstituted pyrilium salts possessing alkyl-, aryl- or combinations of alkyl and aryl groups at the pyridinium ring. The new compounds reported here were assayed for the inhibition of four physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isozymes: the cytosolic hCA I and II, the membrane-anchored bCA IV, and the membrane-bound, tumor-associated isozyme hCA IX. They showed potent inhibitory activity against all investigated isozymes, although with different profiles. For CA I the new derivatives showed inhibition constants in the range of 3-12 nM, for CA II in the range of 0.20-5.96 nM, against CA IV in the range of 2.0-10.3 nM, and against CA IX in the range of 3-45 nM, respectively. These new compounds are membrane-impermeant due to their salt-like character. Some of these derivatives were also tested for their inhibitory activity against the Cl(-)/HCO(3)(-) anion exchanger AE1: two derivatives showed inhibitory activity in the low micromolar range, whereas one compound was inactive at these concentrations. The high affinity of these new derivatives for the tumor-associated isozyme CA IX and their membrane impermeability make this type of CA inhibitor interesting candidates for the selective inhibition of only the tumor-associated isozyme and not the cytosolic ones, for which they also show high potency. Furthermore, we prove here for the first time that the CA-AE metabolon can be inhibited by the same type of sulfonamide derivative.

To allow cells to control their pH and bicarbonate levels, cells express bicarbonate transport proteins that rapidly and selectively move bicarbonate across the plasma membrane. Physical interactions have been identified between the carbonic anhydrase isoform, CAII, and the erythrocyte membrane Cl- /HCO3(-) anion exchanger, AE1, mediated by an acidic motif in the AE1 C-terminus. We have found that the presence of CAII attached to AE1 accelerates AE1 HCO3(-) transport activity, as AE1 moves bicarbonate either into or out of the cell. In efflux mode the presence of CAII attached to AE1 will increase the local concentration of bicarbonate at the AE1 transport site. As bicarbonate is transported into the cell by AE1, the presence of CAII on the cytosolic surface accelerates transport by consumption of bicarbonate, thereby maximizing the transmembrane bicarbonate concentration gradient experienced by the AE1 molecule. Functional and physical interactions also occur between CAII and Na+/HCO3(-) co-transporter isoforms NBC1 and NBC3. All examined bicarbonate transport proteins, except the DRA (SLC26A3) Cl-/HCO3(-) exchange protein, have a consensus CAII binding site in their cytoplasmic C-terminus. Interestingly, CAII does not bind DRA. CAIV is anchored to the extracellular surface of cells via a glycosylphosphatidyl inositol linkage. We have identified extracellular regions of AE1 and NBC1 that directly interact with CAIV, to form a physical complex between the proteins. In summary, bicarbonate transporters directly interact with the CAII and CAIV carbonic anhydrases to increase the transmembrane bicarbonate flux. The complex of a bicarbonate transporter with carbonic anhydrase forms a "Bicarbonate Transport Metabolon."

Previously we have reported that in vitro muscle aldolase binds to muscle FBPase [Biochem. Biophys. Res. Commun. 275 (2000) 611-616] which results in the changes of regulatory properties of the latter enzyme. In the present paper, the evidence that aldolase binds to FBPase in living cell is presented. The colocalization experiment, in which aldolase was diffused into skinned fibres that had been pre-incubated with FBPase, has shown that aldolase in the presence of FBPase binds predominantly to the Z-line. The existence of a triple aldolase-FBPase-alpha-actinin complex was confirmed through a real-time interaction analysis using the BIAcore biosensor. The colocalization of FBPase and aldolase on alpha-actinin of the Z-line indicates the existence of glyconeogenic metabolon in vertebrates' myocytes.

In Saccharomyces cerevisiae, which uses the nonlinear pathway of arginine biosynthesis, the first two enzymes, N-acetylglutamate synthase (NAGS) and N-acetylglutamate kinase (NAGK), are controlled by feedback inhibition. We have previously shown that NAGS and NAGK associate in a complex, essential to synthase activity and protein level [Abadjieva, A., Pauwels, K., Hilven, P. & Crabeel, M. (2001) J. Biol. Chem.276, 42869–42880]. The NAGKs of ascomycetes possess, in addition to the catalytic domain that is shared by all other NAGKs and whose structure has been determined, a C-terminal domain of unknown function and structure. Exploring the role of these two domains in the synthase/kinase interaction, we demonstrate that the ascomycete-specific domain is required to maintain synthase activity and protein level. Previous results had suggested a participation of the third enzyme of the pathway, N-acetylglutamylphosphate reductase, in the metabolon. Here, genetic analyses conducted in yeast at physiological level, or in a heterologous background, clearly demonstrate that the reductase is dispensable for synthase activity and protein level. Most importantly, we show that the arginine feedback regulation of the NAGS and NAGK enzymes is mutually interdependent. First, the kinase becomes less sensitive to arginine feedback inhibition in the absence of the synthase. Second, and as in Neurospora crassa, in a yeast kinase mutant resistant to arginine feedback inhibition, the synthase becomes feedback resistant concomitantly. We conclude that the NAGS/NAGK metabolon promotes the co-ordination of the catalytic activities and feedback regulation of the first two, flux controlling, enzymes of the arginine pathway.