Conceptual Insights, Overview of Carbohydrate and Fatty Acid Metabolism
will help you understand how fatty acid metabolism fits in with other energy storage and utilization pathways (glycolysis, citric acid cyclem, pentose phosphate pathwaym, glycogen metabolism), with a focus on carbon and energy flux.
Fatty acid synthesis is not simply a reversal of the degradative pathway. Rather, it consists of a new set of reactions, again exemplifying the principle that synthetic and degradative pathways are almost always distinct. Some important differences between the pathways are:
Synthesis takes place in the cytosol, in contrast with degradation, which takes place primarily in the mitochondrial matrix.
Intermediates in fatty acid synthesis are covalently linked to the sulfhydryl groups of an acyl carrier protein (ACP), whereas intermediates in fatty acid breakdown are covalently attached to the sulfhydryl group of coenzyme A.
The enzymes of fatty acid synthesis in higher organisms are joined in a single polypeptide chain called fatty acid synthase. In contrast, the degradative enzymes do not seem to be associated.
The growing fatty acid chain is elongated by the sequential addition of two-carbon units derived from acetyl CoA. The activated donor of twocarbon units in the elongation step is malonyl ACP. The elongation reaction is driven by the release of CO2.
The reductant in fatty acid synthesis is NADPH, whereas the oxidants in fatty acid degradation are NAD+ and FAD.
Elongation by the fatty acid synthase complex stops on formation of palmitate (C16). Further elongation and the insertion of double bonds are carried out by other enzyme systems.
22.4.1. The Formation of Malonyl Coenzyme A Is the Committed Step in Fatty Acid Synthesis
Fatty acid synthesis starts with the carboxylation of acetyl CoA to malonyl CoA. This irreversible reaction is the committed step in fatty acid synthesis.
The synthesis of malonyl CoA is catalyzed by acetyl CoA carboxylase, which contains a biotin prosthetic group. The carboxyl group of biotin is covalently attached to the ϵ amino group of a lysine residue, as in pyruvate carboxylase (Section 16.3.2) and propionyl CoA carboxylase (Section 22.3.3). As with these other enzymes, a carboxybiotin intermediate is formed at the expense of the hydrolysis a molecule of ATP. The activated CO2 group in this intermediate is then transferred to acetyl CoA to form malonyl CoA.
This enzyme is also the essential regulatory enzyme for fatty acid metabolism (Section 22.5).
22.4.2. Intermediates in Fatty Acid Synthesis Are Attached to an Acyl Carrier Protein
The intermediates in fatty acid synthesis are linked to an acyl carrier protein. Specifically, they are linked to the sulfhydryl terminus of a phosphopantetheine group, which is, in turn, attached to a serine residue of the acyl carrier protein (Figure 22.21). Recall that, in the degradation of fatty acids, a phosphopantetheine group is present as part of CoA instead (Section 22.2.2). ACP, a single polypeptide chain of 77 residues, can be regarded as a giant prosthetic group, a “macro CoA.”
Phosphopantetheine. Both acyl carrier protein and CoA include phosphopantetheine as their reactive units.
22.4.3. The Elongation Cycle in Fatty Acid Synthesis
The enzyme system that catalyzes the synthesis of saturated long-chain fatty acids from acetyl CoA, malonyl CoA, and NADPH is called the fatty acid synthase. The constituent enzymes of bacterial fatty acid synthases are dissociated when the cells are broken apart. The availability of these isolated enzymes has facilitated the elucidation of the steps in fatty acid synthesis (Table 22.2). In fact, the reactions leading to fatty acid synthesis in higher organisms are very much like those of bacteria.
Principal reactions in fatty acid synthesis in bacteria.
The elongation phase of fatty acid synthesis starts with the formation of acetyl ACP and malonyl ACP. Acetyl transacylase and malonyl transacylase catalyze these reactions.
Malonyl transacylase is highly specific, whereas acetyl transacylase can transfer acyl groups other than the acetyl unit, though at a much slower rate. Fatty acids with an odd number of carbon atoms are synthesized starting with propionyl ACP, which is formed from propionyl CoA by acetyl transacylase.
Acetyl ACP and malonyl ACP react to form acetoacetyl ACP (Figure 22.22). The acyl-malonyl ACP condensing enzyme catalyzes this condensation reaction.
Fatty Acid Synthesis. Fatty acids are synthesized by the repetition of the following reaction sequence: condensation, reduction, dehydration, and reduction. The intermediates shown here are produced in the first round of synthesis.
In the condensation reaction, a four-carbon unit is formed from a twocarbon unit and a three-carbon unit, and CO2 is released. Why is the four-carbon unit not formed from 2 two-carbon units? In other words, why are the reactants acetyl ACP and malonyl ACP rather than two molecules of acetyl ACP? The answer is that the equilibrium for the synthesis of acetoacetyl ACP from two molecules of acetyl ACP is highly unfavorable. In contrast, the equilibrium is favorable if malonyl ACP is a reactant because its decarboxylation contributes a substantial decrease in free energy. In effect, ATP drives the condensation reaction, though ATP does not directly participate in the condensation reaction. Rather, ATP is used to carboxylate acetyl CoA to malonyl CoA. The free energy thus stored in malonyl CoA is released in the decarboxylation accompanying the formation of acetoacetyl ACP. Although HCO3- is required for fatty acid synthesis, its carbon atom does not appear in the product. Rather, all the carbon atoms of fatty acids containing an even number of carbon atoms are derived from acetyl CoA.
The next three steps in fatty acid synthesis reduce the keto group at C-3 to a methylene group (see Figure 22.22). First, acetoacetyl ACP is reduced to d-3-hydroxybutyryl ACP. This reaction differs from the corresponding one in fatty acid degradation in two respects: (1) the d rather than the l isomer is formed; and (2) NADPH is the reducing agent, whereas NAD+ is the oxidizing agent in β oxidation. This difference exemplifies the general principle that NADPH is consumed in biosynthetic reactions, whereas NADH is generated in energy-yielding reactions. Then d-3-hydroxybutyryl ACP is dehydrated to form crotonyl ACP, which is a trans-Δ2-enoyl ACP. The final step in the cycle reduces crotonyl ACP to butyryl ACP. NADPH is again the reductant, whereas FAD is the oxidant in the corresponding reaction in β-oxidation. The enzyme that catalyzes this step, enoyl ACP reductase, is inhibited by triclosan, a broad-spectrum antibacterial agent. Triclosan is used in a variety of products such as toothpaste, soaps, and skin creams. These last three reactions—a reduction, a dehydration, and a second reduction—convert acetoacetyl ACP into butyryl ACP, which completes the first elongation cycle.
In the second round of fatty acid synthesis, butyryl ACP condenses with malonyl ACP to form a C6-β-ketoacyl ACP. This reaction is like the one in the first round, in which acetyl ACP condenses with malonyl ACP to form a C4-β-ketoacyl ACP. Reduction, dehydration, and a second reduction convert the C6-β-ketoacyl ACP into a C6-acyl ACP, which is ready for a third round of elongation. The elongation cycles continue until C16-acyl ACP is formed. This intermediate is a good substrate for a thioesterase that hydrolyzes C16-acyl ACP to yield palmitate and ACP. The thioesterase acts as a ruler to determine fatty acid chain length. The synthesis of longer-chain fatty acids is discussed in Section 22.6.
22.4.4. Fatty Acids Are Synthesized by a Multifunctional Enzyme Complex in Eukaryotes
Although the basic biochemical reactions in fatty acid synthesis are very similar in E. coli and eukaryotes, the structure of the synthase varies considerably. The fatty acid synthases of eukaryotes, in contrast with those of E. coli, have the component enzymes linked in a large polypeptide chain.
Mammalian fatty acid synthase is a dimer of identical 260-kd subunits. Each chain is folded into three domains joined by flexible regions (Figure 22.23). Domain 1, the substrate entry and condensation unit, contains acetyl transferase, malonyl transferase, and β-ketoacyl synthase (condensing enzyme). Domain 2, the reduction unit, contains the acyl carrier protein, β-ketoacyl reductase, dehydratase, and enoyl reductase. Domain 3, the palmitate release unit, contains the thioesterase. Thus, seven different catalytic sites are present on a single polypeptide chain. It is noteworthy that many eukaryotic multienzyme complexes are multifunctional proteins in which different enzymes are linked covalently. An advantage of this arrangement is that the synthetic activity of different enzymes is coordinated. In addition, a multienzyme complex consisting of covalently joined enzymes is more stable than one formed by noncovalent attractions. Furthermore, intermediates can be efficiently handed from one active site to another without leaving the assembly. It seems likely that multifunctional enzymes such as fatty acid synthase arose in eukaryotic evolution by exon shuffling (Section 5.6.2), because each of the component enzymes is recognizably homologous to its bacterial counterpart.
Schematic Representation of Animal Fatty Acid Synthase. Each of the identical chains in the dimer contains three domains. Domain 1 (blue) contains acetyl transferase (AT), malonyl transferase (MT), and condensing enzyme (CE). Domain 2 (yellow) contains (more...)
22.4.5. The Flexible Phosphopantetheinyl Unit of ACP Carries Substrate from One Active Site to Another
We next examine the coordinated functioning of the mammalian fatty acid synthase. Fatty acid synthesis begins with the transfer of the acetyl group of acetyl CoA first to a serine residue in the active site of acetyl transferase and then to the sulfur atom of a cysteine residue in the active site of the condensing enzyme on one chain of the dimeric enzyme. Similarly, the malonyl group is transferred from malonyl CoA first to a serine residue in the active site of malonyl transferase and then to the sulfur atom of the phosphopantetheinyl group of the acyl carrier protein on the other chain in the dimer. Domain 1 of each chain of this dimer interacts with domains 2 and 3 of the other chain. Thus, each of the two functional units of the synthase consists of domains formed by different chains. Indeed, the arenas of catalytic action are the interfaces between domains on opposite chains.
Elongation begins with the joining of the acetyl unit on the condensing enzyme (CE) to a two-carbon part of the malonyl unit on ACP (Figure 22.24).
Reactions of Fatty Acid Synthase. Translocations of the elongating fatty acyl chain between the cysteine sulfhydryl group of the condensing enzyme (CE, blue) and the phosphopantetheine sulfhydryl group of the acyl carrier protein (ACP, yellow) lead to (more...)
CO2 is released and an acetoacetyl-S-phosphopantetheinyl unit is formed on ACP. The active-site sulfhydryl group on the condensing enzyme is restored. The acetoacetyl group is then delivered to three active sites in domain 2 of the opposite chain to reduce it to a butyryl unit. This saturated C4 unit then migrates from the phosphopantetheinyl sulfur atom on ACP to the cysteine sulfur atom on the condensing enzyme. The synthase is now ready for another round of elongation. The butyryl unit on the condensing enzyme becomes linked to a two-carbon part of the malonyl unit on ACP to form a six-carbon unit on ACP, which undergoes reduction. Five more rounds of condensation and reduction produce a palmitoyl (C16) chain on the condensing enzyme, which is hydrolyzed to palmitate by the thioesterase on domain 3 of the opposite chain. The migration of the growing fatty acyl chain back and forth between ACP and the condensing enzyme in each round of elongation is analogous to the translocations of growing peptide chains that take place in protein synthesis (Section 29.3.7).
The flexibility and 20-Å maximal length of the phosphopantetheinyl moiety are critical for the function of this multienzyme complex. The enzyme subunits need not undergo large structural rearrangements to interact with the substrate. Instead, the substrate is on a long, flexible arm that can reach each of the numerous active sites. Recall that biotin and lipoamide also are on long, flexible arms in their multienzyme complexes. The organization of the fatty acid synthases of higher organisms enhances the efficiency of the overall process because intermediates are directly transferred from one active site to the next.
22.4.6. The Stoichiometry of Fatty Acid Synthesis
The stoichiometry of the synthesis of palmitate is
The equation for the synthesis of the malonyl CoA used in the preceding reaction is
Hence, the overall stoichiometry for the synthesis of palmitate is
22.4.7. Citrate Carries Acetyl Groups from Mitochondria to the Cytosol for Fatty Acid Synthesis
The synthesis of palmitate requires the input of 8 molecules of acetyl CoA, 14 molecules of NADPH, and 7 molecules of ATP. Fatty acids are synthesized in the cytosol, whereas acetyl CoA is formed from pyruvate in mitochondria. Hence, acetyl CoA must be transferred from mitochondria to the cytosol. Mitochondria, however, are not readily permeable to acetyl CoA. Recall that carnitine carries only long-chain fatty acids. The barrier to acetyl CoA is bypassed by citrate, which carries acetyl groups across the inner mitochondrial membrane. Citrate is formed in the mitochondrial matrix by the condensation of acetyl CoA with oxaloacetate (Figure 22.25). When present at high levels, citrate is transported to the cytosol, where it is cleaved by ATP-citrate lyase.
Enzymes catalyzing the cleavage of C-C, C-O, or C-N bonds by elimination. A double bond is formed in these reactions.
Transfer of Acetyl CoA to the Cytosol. Acetyl CoA is transferred from mitochondria to the cytosol, and the reducing potential NADH is concomitantly converted into that of NADPH by this series of reactions.
Thus, acetyl CoA and oxaloacetate are transferred from mitochondria to the cytosol at the expense of the hydrolysis of a molecule of ATP.
22.4.8. Sources of NADPH for Fatty Acid Synthesis
Oxaloacetate formed in the transfer of acetyl groups to the cytosol must now be returned to the mitochondria. The inner mitochondrial membrane is impermeable to oxaloacetate. Hence, a series of bypass reactions are needed. Most important, these reactions generate much of the NADPH needed for fatty acid synthesis. First, oxaloacetate is reduced to malate by NADH. This reaction is catalyzed by a malate dehydrogenase in the cytosol.
Second, malate is oxidatively decarboxylated by an NADP+-linked malate enzyme (also called malic enzyme).
The pyruvate formed in this reaction readily enters mitochondria, where it is carboxylated to oxaloacetate by pyruvate carboxylase.
The sum of these three reactions is
Thus, one molecule of NADPH is generated for each molecule of acetyl CoA that is transferred from mitochondria to the cytosol. Hence, eight molecules of NADPH are formed when eight molecules of acetyl CoA are transferred to the cytosol for the synthesis of palmitate. The additional six molecules of NADPH required for this process come from the pentose phosphate pathway (Section 20.3.1).
The accumulation of the precursors for fatty acid synthesis is a wonderful example of the coordinated use of multiple processes to fulfill a biochemical need. The citric acid cycle, subcellular compartmentalization, and the pentose phosphate pathway provide the carbon atoms and reducing power, whereas glycolysis and oxidative phosphorylation provide the ATP to meet the needs for fatty acid synthesis.
22.4.9. Fatty Acid Synthase Inhibitors May Be Useful Drugs
Fatty acid synthase is overexpressed in some breast cancers. Researchers intrigued by this observation have tested inhibitors of fatty acid synthase on mice to see how the inhibitors affect tumor growth. A startling observation was made: mice treated with inhibitors of the condensing enzyme showed remarkable weight loss due to inhibition of feeding. The results of additional studies revealed that this inhibition is due, at least in part, to the accumulation of malonyl CoA. Thus, fatty acid synthase inhibitors are exciting candidates both as antitumor and as antiobesity drugs.
22.4.10. Variations on a Theme: Polyketide and Nonribosomal Peptide Synthetases Resemble Fatty Acid Synthase
The mammalian multifunctional fatty acid synthase is a member of a large family of complex enzymes termed megasynthases that participate in step-by-step synthetic pathways. Two important classes of compounds that are synthesized by such enzymes are the polyketides and the nonribosomal peptides. The antibiotic erythromycin is an example of a polyketide, whereas penicillin (Section 8.5.5) is a nonribosomal peptide.
The core of erythromycin (deoxyerythronolide B, or Deb) is synthesized by the following reaction:
This reaction is accomplished by three megasynthases consisting of 3491, 3567, and 3172 amino acids. The synthesis of deoxyerythronolide B begins with propionyl CoA linked to a phosphopantetheine chain connected to an acyl carrier protein domain. Similarly, the precursor of penicillin [Δ-(l-aminoadipyl)-l-cysteinyl-d-valine, or ACV] is generated by the following reaction:
which is catalyzed by a megasynthase consisting of 3791 amino acids. Each amino acid is activated by a specific adenylation domain within the enzyme—a domain that is homologous to acyl CoA synthase. Additional domains are responsible for peptide-bond formation and for the epimerization of the valine residue. Again, during synthesis, the components are linked to phosphopantetheine chains. Members of this remarkably modular enzyme family generate many of the natural products that have proved to be useful as drugs.
Acetyl-CoA Synthase (ACS), not to be confused with Acetyl-CoA Synthetase or Acetate-CoA Ligase (ADP forming), is a Nickel containing enzyme involved in the metabolic processes of cells. Together with Carbon monoxide dehydrogenase (CODH), it forms the bifunctional enzyme Acetyl-CoA Synthase/Carbon Monoxide Dehydrogenase (ACS/CODH) found in anaerobic organisms such as archaea and bacteria. The ACS/CODH enzyme works primarily through the Wood–Ljungdahl pathway which converts Carbon dioxide to Acetyl-CoA. The recommended name for this enzyme is CO-methylating acetyl-CoA synthase.
In nature, there are six different pathways where CO2 is fixed. Of these, the Wood–Ljungdahl pathway is the predominant sink in anaerobic conditions. Acetyl-CoA Synthase (ACS) and carbon monoxide dehydrogenase (CODH) are integral enzymes in this one pathway and can perform diverse reactions in the carbon cycle as a result. Because of this, the exact activity of these molecules has come under intense scrutiny over the past decade.
The Wood–Ljungdahl pathway consists of two different reactions that break down carbon dioxide. The first pathway involves CODH converting carbon dioxide into carbon monoxide through a two-electron transfer, and the second reaction involves ACS synthesizing acetyl-CoA using the carbon monoxide from CODH together with coenzyme-A (CoA) and a methyl group from a corrinoid-iron sulfur protein, CFeSP. The two main overall reactions are as follows:
The Acetyl-CoA produced can be used in a variety of ways depending on the needs of the organism. For example, acetate-forming bacteria use acetyl-CoA for their autotrophic growth processes, and methanogenic archae such as Methanocarcina barkeri convert the acetyl-CoA into acetate and use it as an alternative source of carbon instead of CO2.
Since the above two reactions are reversible, it opens up a diverse range of reactions in the carbon cycle. In addition to acetyl-CoA production, the reverse can occur with ACS producing acetate, CO and returning the methyl piece back to the corrinoid protein. Acetogenic bacteria use this method to generate acetate and acetic acid. Along with the process of methanogenesis, organisms can subsequently convert the acetate to methane. Furthermore, the Wood-Ljungdahl pathway allows for the anaerobic oxidation of acetate where ATP is used to convert acetate into acetyl-CoA, which is then broken down by ACS to produce carbon dioxide that is released into the atmosphere.
It has been discovered that the CODH/ACS enzyme in the bacteria M. theroaceticum can make dinitrogen (N2) from nitrous oxide in the presence of an electron-donating species. It can also catalyze the reduction of the pollutant, 2,4,6-trinitrotoluene (TNT) and catalyze the oxidation of n-butyl isocyanide.
The first, and one of the most comprehensive, crystal structures of ACS/CODH from the bacteria M. thermoacetica was presented in 2002 by Drennan and colleagues. In this paper they constructed a heterotetramer, with the active site "A-cluster" residing in the ACS subunit and the active site "C-cluster" in CODH subunit. Furthermore, they resolved the structure of the A-cluster active site and found an [Fe4S4]-X-Cu-X-Ni centre which is highly unusual in biology. This structural representation consisted of a [Fe4S4] unit bridged to a binuclear centre, where Ni(II) resided in the distal position (denoted as Nid) in a square-planar conformation and a Cu(I) ion resided in the proximal position in a distorted tetrahedral position with ligands of unknown identity.
The debate towards the absolute structure and identity of the metals in the A-cluster active site of ACS continued, with a competing model presented. The authors suggested two different forms of the ACS enzyme, an "Open" form and a "Closed" form, with different metals occupying the proximal metal site (denoted as Mp) for each form. The general scheme of the enzyme followed closely with the first study's findings, but this new structure proposed a Nickel ion in the "open" form and a Zinc ion in the "closed" form.
A later review article attempted to reconcile the different observations of Mp and stated that this proximal position in the active site of ACS was prone to substitution and could contain any one of Cu, Zn and Ni. The three forms of this A-cluster most likely hold a small amount of Ni and a relatively larger amount of Cu.
Present (2014 onwards)
It is now generally accepted that the ACS active site (A-cluster) is a Ni-Ni metal centre with both nickels having a +2 oxidation state. The [Fe4S4] cluster is bridged to the closer nickel, Np which is connected via a thiolate bridge to the farther nickel, Nid. Nid is coordinated to two cysteine molecules and two backbone amide compounds, and is in a square-planar coordination. The space next to the metal can accommodate substrates and products. Nip is in a T-shaped environment bound to three sulfur atoms, with an unknown ligand possibly creating a distorted tetrahedral environment. This ligand has been hypothesized to be a water molecule or an acetyl group in the surrounding area in the cell. Although the proximal nickel is labile and can be replaced with a Cu of Zn centre, experimental evidence suggests that activity of ACS is limited to the presence of nickel only. In addition, some studies have shown that copper can even inhibit the enzyme under certain conditions.
The overall structure of the CODH/ACS enzyme consists of the CODH enzyme as a dimer at the centre with two ACS subunits on each side. The CODH core is made up of two Ni-Fe-S clusters (C-cluster), two [Fe4S4] clusters (B-cluster) and one [Fe4S4] D-cluster. The D-cluster bridges the two subunits with one C and one B cluster in each monomer, allowing rapid electron transfer. The A-cluster of ACS is in constant communication with the C-cluster in CODH. This active site is also responsible for the C-C and C-S bond formations in the product acetyl-CoA (and its reverse reaction).
The ACS enzyme contains three main subunits. The first is the active site itself with the NiFeS centre. The second is the portion that directly interacts with CODH in the Wood-Ljungdahl pathway. This part is made up of α-helices that go into a Rossman fold. It also appears to interact with a ferredoxin compound which may activate the subunit during the CO transferring process from CODH to ACS. The final domain binds CoA and consists of six arginine residues with a tryptophan molecule.
Experiments between the C-cluster of CODH and the A-cluster of ACS reveal a long, hydrophobic channel connecting the two domains to allow for the transfer of carbon monoxide from CODH to ACS. This channel is most likely to protect the carbon monoxide molecules from the outside environment of the enzyme and to increase efficiency of acetyl-CoA production.
Studies in literature have been able to isolate the CODH/ACS enzyme in an "open" and "closed" configuration. This has led to the hypothesis that it undergoes four conformational changes depending on its activity. With the "open" position, the active site rotates itself to interact with the CFeSP protein in the methyl transfer step of the Wood-Ljungdahl pathway. The "closed" position opens up the channel between CODH and ACS to allow for the transfer of CO. These two configurations are opposite one another in that access to CO blocks off interaction with CFeSP, and when methylation occurs, the active site is buried and does not allow CO transfer. A second "closed" position is needed to block off water from the reaction. Finally, the A-cluster must be rotated once more to allow for the binding of CoA and release of the product. The exact trigger of these structural changes and the mechanistic details have yet to be resolved.
Two competing mechanisms have been proposed for the formation of acetyl-CoA, the "Paramagnetic mechanism" and the "Diamagnetic mechanism". Both are similar in terms of the binding of substrates and the general steps, but differ in the oxidation state of the metal centre. Nip is believed to be the substrate binding centre which undergoes redox. The farther nickel centre and the [Fe4S4] cluster are not thought to be involved in the process.
In the paramagnetic mechanism, some type of complex (ferrodoxin, for example) activates the Nip atom, reducing it from Ni2+ to Ni1+. The nickel then binds to either carbon monoxide from CODH or the methyl group donated by the CFeSP protein in no particular order. This is followed by migratory insertion to form an intermediate complex. CoA then binds to the metal and the final product, acetyl-CoA, is formed. Some criticisms of this mechanism are that it is unbalanced in terms of electron count and the activated Ni+1 intermediate cannot be detected with electron paramagnetic resonance. Furthermore, there is evidence of the ACS catalytic cycle without any external reducing complex, which refutes the ferrodoxin activation step.
The second proposed mechanism, the diamagnetic mechanism, involves a Ni0 intermediate instead of a Ni1+. After addition of the methyl group and carbon monoxide, followed by insertion to produce the metal-acetyl complex, CoA attacks to produce the final product. The order in which the carbon monoxide molecule and the methyl group bind to the nickel centre has been highly debated, but no solid evidence has demonstrated preference for one over the other. Although this mechanism is electronically balanced, the idea of a Ni0 species is highly unprecedented in biology. There has also been no solid evidence supporting the presence of a zero-valent Ni species. However, similar nickel species to ACS with a Ni0 centre have been made, so the diamagnetic mechanism is not an implausible hypothesis.
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