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Transketolase
Transketolase
Transketolase
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transketolase
Identifiers
EC no.2.2.1.1
CAS no.9014-48-6
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BRENDABRENDA entry
ExPASyNiceZyme view
KEGGKEGG entry
MetaCycmetabolic pathway
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PDB structuresRCSB PDB PDBe PDBsum
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transketolase
Identifiers
SymbolTKT
NCBI gene7086
HGNC11834
OMIM606781
RefSeqNM_001064
UniProtP29401
Other data
EC number2.2.1.1
LocusChr. 3 p14.3
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StructuresSwiss-model
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Transketolase (abbreviated as TK) is an enzyme that, in humans, is encoded by the TKT gene.[1] It participates in both the pentose phosphate pathway in all organisms and the Calvin cycle of photosynthesis. Transketolase catalyzes two important reactions, which operate in opposite directions in these two pathways. In the first reaction of the non-oxidative pentose phosphate pathway, the cofactor thiamine diphosphate accepts a 2-carbon fragment from a 5-carbon ketose (D-xylulose-5-P), then transfers this fragment to a 5-carbon aldose (D-ribose-5-P) to form a 7-carbon ketose (sedoheptulose-7-P). The abstraction of two carbons from D-xylulose-5-P yields the 3-carbon aldose glyceraldehyde-3-P. In the Calvin cycle, transketolase catalyzes the reverse reaction, the conversion of sedoheptulose-7-P and glyceraldehyde-3-P to pentoses, the aldose D-ribose-5-P and the ketose D-xylulose-5-P.

The second reaction catalyzed by transketolase in the pentose phosphate pathway involves the same thiamine diphosphate-mediated transfer of a 2-carbon fragment from D-xylulose-5-P to the aldose erythrose-4-phosphate, affording fructose 6-phosphate and glyceraldehyde-3-P. Again, the same reaction occurs in the Calvin cycle but in the opposite direction. Moreover, in the Calvin cycle, this is the first reaction catalyzed by transketolase rather than the second.

Transketolase connects the pentose phosphate pathway to glycolysis, feeding excess sugar phosphates into the main carbohydrate metabolic pathways in mammals. Its presence is necessary for the production of NADPH, especially in tissues actively engaged in biosyntheses, such as fatty acid synthesis by the liver and mammary glands, and for steroid synthesis by the liver and adrenal glands. Thiamine diphosphate is an essential cofactor, along with calcium.

Transketolase is abundantly expressed in the mammalian cornea by the stromal keratocytes and epithelial cells and is reputed to be one of the corneal crystallins.[2]

Species distribution

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Transketolase is widely expressed in many organisms, including bacteria, plants, and mammals. The following human genes encode proteins with transketolase activity: [citation needed]

  • TKT (transketolase)
  • TKTL1 (transketolase-like protein 1)
  • TKTL2 (transketolase-like protein 2)

Structure

[edit]

The entrance to the active site for this enzyme is made up mainly of several arginine, histidine, serine, and aspartate side-chains, with a glutamate side-chain playing a secondary role. These side-chains, specifically Arg359, Arg528, His469, and Ser386, are conserved within each transketolase enzyme and interact with the phosphate group of the donor and acceptor substrates. Because the substrate channel is so narrow, the donor and acceptor substrates cannot be bound simultaneously. Also, the substrates conform into a slightly extended form upon binding in the active site to accommodate this narrow channel.[citation needed]

Although this enzyme can bind numerous types of substrates, such as phosphorylated and nonphosphorylated monosaccharides including the keto and aldosugars fructose, ribose, etc., it has a high specificity for the stereoconfiguration of the hydroxyl groups of the sugars. These hydroxyl groups at C-3 and C-4 of the ketose donor must be in the D-threo configuration to correctly correspond to the C-1 and C-2 positions on the aldose acceptor.[3] Also, they stabilize the substrate in the active site by interacting with the Asp477, His30, and His263 residues. Disruption of this configuration, both the placement of hydroxyl groups or their stereochemistry, would consequently alter the H-bonding between the residues and substrates thus causing a lower affinity for the substrates.[citation needed]

In the first half of this pathway, His263 is used to effectively abstract the C3 hydroxyl proton, which thus allows a 2-carbon segment to be cleaved from fructose 6-phosphate.[4] The cofactor necessary for this step to occur is thiamin pyrophosphate (TPP). The binding of TPP to the enzyme incurs no major conformational change to the enzyme; instead, the enzyme has two flexible loops at the active site that make TPP accessible and binding possible.[3] Thus, this allows the active site to have a "closed" conformation rather than a large conformational change. Later in the pathway, His263 is used as a proton donor for the substrate acceptor-TPP complex, which can then generate erythrose-4-phosphate.[citation needed]

The histidine and aspartate side-chains are used to effectively stabilize the substrate within the active site and participate in deprotonation of the substrate. To be specific, the His 263 and His30 side-chains form hydrogen bonds to the aldehyde end of the substrate, which is deepest into the substrate channel, and Asp477 forms hydrogen bonds with the alpha hydroxyl group on the substrate, where it works to effectively bind the substrate and check for proper stereochemistry. It is also thought that Asp477 could have important catalytic effects because of its orientation in the middle of the active site and its interactions with the alpha hydroxyl group of the substrate. Glu418, located in the deepest region of the active site, plays a critical role in stabilizing the TPP cofactor. Specifically, it is involved in the cofactor-assisted proton abstraction from the substrate molecule.[3]

The phosphate group of the substrate also plays an important role in stabilizing the substrate upon its entrance into the active site. The tight ionic and polar interactions between this phosphate group and the residues Arg359, Arg528, His469, and Ser386 collectively work to stabilize the substrate by forming H-bonds to the oxygen atoms of the phosphate.[3] The ionic nature is found in the salt bridge formed from Arg359 to the phosphate group.[citation needed]

Mechanism

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The catalysis of this mechanism is initiated by the deprotonation of TPP at the thiazolium ring. This carbanion then binds to the carbonyl of the donor substrate, thus cleaving the bond between C-2 and C-3. This keto fragment remains covalently bound to the C-2 carbon of TPP. The donor substrate is then released, and the acceptor substrate enters the active site where the fragment, bound to the intermediate α-β-dihydroxyethyl thiamin diphosphate, is transferred to the acceptor.[3]

Mechanism of fructose-6-phosphate to xylulose-5-phosphate in transketolase active site

Experiments have also been conducted that test the effect of replacing alanine for the amino acids at the entrance to the active site, Arg359, Arg528, and His469, which interact with the phosphate group of the substrate. This replacement creates a mutant enzyme with impaired catalytic activity.[3]

Role in disease

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Transketolase activity decreases due to thiamine deficiency, generally due to malnutrition. Several diseases are associated with thiamine deficiency, including beriberi, biotin-thiamine-responsive basal ganglia disease (BTBGD),[5] Wernicke–Korsakoff syndrome, and others (see thiamine for a comprehensive listing).

In Wernicke–Korsakoff syndrome, while no mutations could be demonstrated,[6] there is an indication that thiamine deficiency leads to Wernicke–Korsakoff syndrome only in those whose transketolase has a reduced affinity for thiamine.[7] In this way, the activity of transketolase is greatly hindered, and, as a consequence, the entire pentose phosphate pathway is inhibited.[8]

In Transketolase Deficiency, also known as SDDHD (Short Stature, Developmental Delay, and congenital Heart Defects), the disease is caused by an inherited autosomal recessive mutation in the TKT gene. A rare disorder of pentose phosphate metabolism with symptoms apparent in infancy including developmental delay and intellectual disability, delayed or absent speech, short stature, and congenital heart defects. Additional reported features include hypotonia, hyperactivity, stereotypic behavior, ophthalmologic abnormalities, hearing impairment, and variable facial dysmorphism, among others. Laboratory analysis shows elevated plasma and urinary polyols (erythritol, arabitol, and ribitol) and urinary sugar-phosphates (ribose-5-phosphate and xylulose/ribulose-5-phosphate).[9] "Cell extracts from all 5 patients showed absent or low residual TKT activity. Boyle et al. (2016) suggested that the low TKT activity in some tissues, possibly from another protein with the same function, might explain why TKT deficiency is compatible with life even though TKT is an essential enzyme."[10]

Diagnostic use

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Red blood cell transketolase (aka ETK for erythrocyte transketolase)[11] activity is reduced in deficiency of thiamine (vitamin B1), and may be used in the diagnosis of Wernicke encephalopathy and other B1-deficiency syndromes if the diagnosis is in doubt.[12] Apart from the baseline enzyme activity (which may be normal even in deficiency states), acceleration of enzyme activity after the addition of thiamine pyrophosphate may be diagnostic of thiamine deficiency (0-15% normal, 15-25% deficiency, >25% severe deficiency).[13]

References

[edit]
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Transketolase

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Transketolase is a thiamine diphosphate (ThDP)-dependent that catalyzes the reversible transfer of a two-carbon ketol unit from donor substrates to acceptor substrates, playing a central role in the non-oxidative branch of the (). This pathway interconnects , enabling the interconversion of sugars to produce ribose-5-phosphate for nucleotide synthesis and NADPH for reductive and defense. In cellular , transketolase facilitates the shunting of excess intermediates into the PPP, balancing the production of pentoses and hexoses while linking it to and other pathways. The operates via a ping-pong mechanism, forming a dihydroxyethyl-ThDP intermediate that transfers the ketol group, and exhibits half-of-the-sites reactivity in its dimeric structure, where active sites alternate between subunits. Structurally, it is a homodimer with each containing ThDP and a divalent cation like Mg²⁺, and its activity is modulated by states, with oxidation enhancing function during . Across species, transketolases share a conserved but vary in details, such as the five-residue histidyl crown in transketolase that distinguishes it from microbial counterparts. Clinically, transketolase is implicated in disorders, particularly Wernicke-Korsakoff syndrome, where reduced enzyme activity due to low levels contributes to neurological damage from impaired PPP function and energy metabolism. Overexpression of transketolase-like isoforms, such as TKTL1, has been associated with cancer progression, highlighting its potential as a therapeutic target. Additionally, deficiencies or abnormalities in transketolase activity are linked to neurodegenerative diseases, , and alcoholism-related brain damage, underscoring its broad physiological importance.

Overview

Definition and Function

Transketolase (EC 2.2.1.1) is a transferase enzyme that catalyzes the reversible transfer of a two-carbon ketol unit, specifically , from donor substrates to acceptor substrates, thereby facilitating the interconversion of sugars in . This enzyme requires (TPP) as an essential cofactor to form a covalent intermediate during the transfer process. In its primary biological role, transketolase contributes to balancing carbohydrate metabolism within the non-oxidative branch of the pentose phosphate pathway (PPP), where it interconverts phosphorylated sugars to maintain equilibrium between glycolytic intermediates and pentose phosphates needed for biosynthetic processes. This activity indirectly supports NADPH generation through the oxidative PPP and provides precursors such as ribose-5-phosphate for nucleotide synthesis, ensuring cellular redox balance and growth. A representative reaction catalyzed by transketolase is the transfer between xylulose 5-phosphate and ribose 5-phosphate: Xylulose 5-phosphate+Ribose 5-phosphate⇌Sedoheptulose 7-phosphate+Glyceraldehyde 3-phosphate\text{Xylulose 5-phosphate} + \text{Ribose 5-phosphate} \rightleftharpoons \text{Sedoheptulose 7-phosphate} + \text{Glyceraldehyde 3-phosphate} Transketolase was discovered in the early 1950s during the elucidation of the pentose phosphate pathway, with independent identifications by Efraim Racker and Bernard L. Horecker, the latter's group contributing key experiments on its activity with sugar phosphates. Their work, including purification and characterization from yeast extracts, established transketolase as a critical component of non-oxidative carbon shuffling in metabolism.

Gene and Expression

The human transketolase enzyme is encoded by the TKT gene, located on chromosome 3p21.31. This gene spans approximately 31 kb of genomic DNA and comprises 15 exons that generate multiple transcript variants, with the primary isoform encoding a 623-amino acid protein. The calculated molecular weight of this polypeptide is approximately 67.5 kDa, consistent with its role as a key metabolic enzyme. TKT mRNA expression is detectable across a broad array of tissues, reflecting the enzyme's ubiquitous involvement in cellular , but it exhibits tissue-specific patterns of abundance. from the Genotype-Tissue Expression (GTEx) and the Human Protein Atlas indicate elevated levels in metabolically demanding tissues such as the liver, , , and , where transcript per million (TPM) values often exceed 20-50. In contrast, expression is notably lower in the (TPM ~5-10) and (TPM ~3-8), underscoring differential reliance on flux in these organs. The TKT gene demonstrates strong evolutionary conservation, with orthologs present in prokaryotes and eukaryotes alike, highlighting its ancient origins tied to fundamental carbohydrate metabolism. In bacteria, such as Escherichia coli, two homologous genes (tktA and tktB) encode transketolase isozymes essential for pentose utilization and aromatic compound biosynthesis. Yeast (Saccharomyces cerevisiae) possesses TKL1 and TKL2, while plants like Arabidopsis thaliana have multiple TKT paralogs supporting photosynthetic carbon partitioning. Mammalian homologs, including human TKT, share sequence identity of approximately 25-30% with non-mammalian counterparts, evolving slowly as evidenced by molecular clock analyses. Regulation of TKT expression is responsive to nutritional cues, particularly glucose availability, which promotes upregulation via insulin signaling pathways. Insulin acts through the to enhance TKT transcription, often in coordination with sterol regulatory element-binding protein (SREBP) family members that integrate glucose and lipid metabolic signals in the liver and . This mechanism ensures adaptive flux through the non-oxidative during abundance.

Structure and Biochemistry

Protein Structure

Transketolase in eukaryotes functions as a homodimer, with each subunit having a molecular mass of approximately 68 kDa in humans and around 70-74 kDa in yeast. The enzyme's quaternary structure features two identical subunits arranged with C2 symmetry, where the active sites are positioned at the interface between the subunits, ensuring cooperative binding and catalysis. This dimeric assembly is essential for stability and function, as dissociation leads to loss of activity. Each adopts a V-shaped α/β fold composed of three distinct domains connected by flexible linker regions. The N-terminal domain (residues 1–276 in humans), also known as the PP domain, is primarily responsible for substrate binding and contains a Rossmann-like motif that accommodates the moiety of the cofactor. The middle domain (residues 316–472), or Pyr domain, interfaces with the cofactor's aminopyrimidine ring and contributes to the architecture. The C-terminal domain (residues 493–623) plays a regulatory role, though its precise function remains less defined, and stabilizes the overall structure through interactions with the other domains. In , the domain boundaries are similar but extended, with the N-terminal domain spanning residues 3–322, the middle 323–538, and the C-terminal 539–680. The central β-sheets in each domain form a scaffold that supports the enzyme's conformational rigidity. The crystal structure of transketolase was first resolved for the yeast enzyme (Saccharomyces cerevisiae) in 1992 at 2.5 Å resolution, revealing the α/β architecture and cofactor binding site; subsequent refinements improved resolution to 2.0 Å (PDB: 1TRK). The human structure was determined in 2010 at 1.75 Å resolution (PDB: 3MOS), confirming high conservation with yeast (RMSD ~2.1 Å) and highlighting subtle adaptations in the active site cleft. These structures demonstrate a conserved subunit interface dominated by a hydrophobic core involving residues such as Val380 and leucine/ valine clusters from both subunits, which mediate non-covalent interactions critical for dimerization. Dimer stability is influenced by -dependent conformational changes, particularly in the cofactor-binding loops and interface regions. At neutral to slightly alkaline , the maintains a compact dimeric form, but deviations—such as acidification—can induce partial unfolding and aggregation, reducing stability without full dissociation. These changes are analogous across , with eukaryotic transketolase showing enhanced dimer integrity in the presence of cofactors at physiological .

Cofactors and Active Site

Transketolase requires (TPP) as its primary cofactor, which binds non-covalently in a V-shaped conformation at the to facilitate the transfer of a two-carbon ketol unit between substrates. The TPP binding is stabilized by a divalent metal , typically Mg²⁺ (physiological), which coordinates to the diphosphate moiety of TPP and specific protein residues, including Asp155, Asn185, and the backbone carbonyl of Leu187, enabling the cofactor's role in generation. While Mg²⁺ is the physiological cation, Ca²⁺ is often used in and coordinates similarly. This metal coordination is essential for the holoenzyme formation, as the apo form lacks TPP and exhibits an open, disordered with flexible loops that prevent substrate access. The of human transketolase features conserved residues critical for cofactor stabilization and substrate interaction, including Arg359, which positions the substrate by binding its group, and His469, involved in proton from the donor substrate. Glu366 (in the Pyr domain) plays a key role in stabilizing the intermediate of TPP by forming a with the N1′ nitrogen of the ring, promoting deprotonation at the C2 position of the thiazolium ring. These residues, along with Asp475, which coordinates interactions with substrate hydroxyl groups, are highly conserved across and contribute to the enzyme's specificity for ketol transfer. Substrate specificity in transketolase is determined by a hydrophobic pocket in the , lined with aromatic residues such as Phe and Tyr, which preferentially accommodates D-erythro and D-threo configurations at the C3-C4 positions of donor ketoses, ensuring stereoselective recognition and binding. Upon TPP binding, the undergoes a conformational closure, organizing the cofactor-binding loops and creating a symmetric environment in the dimeric holoenzyme that enhances catalytic efficiency compared to the open apo form. This transition from apo to holo state is quasi-irreversible in mammalian transketolases, underscoring the cofactor's role in maintaining structural integrity and function. Recent comparative structural studies (as of 2024) highlight conserved folds across species but note variations in loops that influence substrate preferences in microbial transketolases.

Mechanism of Action

Reactions Catalyzed

Transketolase catalyzes the reversible transfer of a two-carbon ketol unit from a donor to an acceptor in the non-oxidative phase of the (PPP). The primary reactions facilitate the interconversion of sugar phosphates, enabling the pathway to generate glycolytic intermediates or pentose sugars for biosynthesis depending on cellular needs. These reactions are essential for balancing carbon flux between the PPP and . The first key reaction involves the donor D-xylulose 5-phosphate and the acceptor D-ribose 5-phosphate, yielding D-sedoheptulose 7-phosphate and D-glyceraldehyde 3-phosphate: D-Xylulose 5-phosphate+D-Ribose 5-phosphate⇌D-Sedoheptulose 7-phosphate+D-Glyceraldehyde 3-phosphate\text{D-Xylulose 5-phosphate} + \text{D-Ribose 5-phosphate} \rightleftharpoons \text{D-Sedoheptulose 7-phosphate} + \text{D-Glyceraldehyde 3-phosphate} This step, identified in early biochemical studies of the PPP, supports the production of seven-carbon sugars for subsequent transaldolase action.33105-1/fulltext) The second primary reaction uses D-xylulose 5-phosphate as the donor with D-erythrose 4-phosphate as the acceptor, producing D-fructose 6-phosphate and D-glyceraldehyde 3-phosphate: D-Xylulose 5-phosphate+D-Erythrose 4-phosphate⇌D-Fructose 6-phosphate+D-Glyceraldehyde 3-phosphate\text{D-Xylulose 5-phosphate} + \text{D-Erythrose 4-phosphate} \rightleftharpoons \text{D-Fructose 6-phosphate} + \text{D-Glyceraldehyde 3-phosphate} This reaction completes the non-oxidative branch by generating glycolytic intermediates, allowing excess pentoses to feed into central metabolism.33105-1/fulltext) These reactions are highly reversible, with equilibrium constants (K_eq) near unity—for the first reaction, K_eq ≈ 0.48 ([Rib5P][Xul5P]/[Sed7P][GAP])—ensuring directionality is dictated by substrate concentrations and metabolic demands rather than thermodynamic favoritism. An additional physiological reaction operates in the reverse direction of the second, converting D-fructose 6-phosphate and D-glyceraldehyde 3-phosphate to D-xylulose 5-phosphate and D-erythrose 4-phosphate, which can replenish PPP intermediates during gluconeogenic conditions. Beyond canonical metabolism, transketolase has been engineered in for asymmetric synthesis of , leveraging its to produce enantiopure derivatives through carboligation of non-phosphorylated substrates.

Step-by-Step Catalysis

Transketolase catalyzes the reversible transfer of a two-carbon ketol unit via a (TPP)-dependent mechanism, where TPP is first activated to form a nucleophilic . In the human enzyme, at the C2 position of the TPP thiazolium ring is mediated by Glu418, generating the that initiates the reaction by to the of the donor substrate. The proceeds in four main steps. First, the donor , such as D-xylulose 5-phosphate, binds near the , coordinated by residues including His30 and His481; the TPP then adds to the C2 carbonyl, forming a covalent ketol-TPP and stabilizing the intermediate through protonation by nearby residues. Second, a retro-aldol cleavage is triggered by of the C3 hydroxyl group (facilitated by His263), breaking the C3-C4 bond in the donor and releasing the product, such as , while forming an -bound glycolaldehyde-TPP intermediate. Third, the acceptor , such as , binds, and the enamine carbon of the glycolaldehyde-TPP attacks the carbonyl of the acceptor, followed by proton transfer to reconstruct the ketol group on the new seven-carbon chain. Finally, the product, such as sedoheptulose 7-phosphate, is released, and Glu418 the TPP to regenerate the , completing the cycle. Kinetic studies of the human enzyme indicate a KmK_m for xylulose 5-phosphate of approximately 0.25 mM and a VmaxV_{max} of about 1.2 μ\mumol/min/mg under standard assay conditions, with optimal activity near pH 7.6. Isotope labeling experiments using 14^{14}C-labeled donor substrates, such as specifically labeled xylulose, have confirmed that the two-carbon glycolaldehyde unit is transferred intact, with no evidence of internal C-C bond breakage within the unit, as the labeling patterns in products directly correspond to the expected retention of the donor's C1-C2 fragment.57048-7/fulltext)

Biological Roles

Distribution Across Species

Transketolase is a ubiquitous enzyme present across all domains of life, including bacteria, archaea, yeast, plants, and animals, reflecting its essential role in carbon metabolism. In prokaryotes such as Escherichia coli, two isoenzymes encoded by the tktA and tktB genes facilitate glycolytic flux and the non-oxidative pentose phosphate pathway, with the double mutant exhibiting auxotrophy for aromatic amino acids and pyridoxine. In eukaryotes, unicellular organisms like the yeast Saccharomyces cerevisiae express a single transketolase isoform from the TKL1 gene, which is critical for efficient glycolysis and aromatic amino acid biosynthesis. Multicellular eukaryotes, including mammals, feature multiple isoforms resulting from gene duplications during vertebrate evolution. In , transketolase exists in distinct chloroplastic and cytosolic forms, both nuclear-encoded, with the chloroplastic isoform comprising over 75% of total activity in photosynthetic tissues and supporting the , while the cytosolic form aids general . The enzyme's sequence exhibits moderate conservation across kingdoms, with human transketolase sharing approximately 27% identity with its counterparts in E. coli (tktA), , and , particularly in catalytic domains. A key conserved feature is the (TPP)-binding motif, characterized by the GDG sequence followed by a variable linker and ending in a conserved , which is preserved in transketolases from to humans. Phylogenetic analyses indicate that transketolase homologs cluster by domain but show evidence of lateral gene transfers in some eukaryotic lineages, such as chlamydial origins in certain photosynthetic protists, underscoring its ancient and adaptable evolutionary history. Overall, this broad distribution highlights transketolase's fundamental conservation for interconverting sugars in diverse metabolic contexts.

Roles in Metabolic Pathways

Transketolase serves as a key in the non-oxidative branch of the (PPP), facilitating the reversible transfer of two-carbon units from donors to acceptors, thereby shunting carbon atoms from excess phosphates to glycolytic intermediates such as fructose-6-phosphate and glyceraldehyde-3-phosphate. This carbon redistribution enables cells to generate ribose-5-phosphate for and while integrating PPP outputs with for energy production. In doing so, transketolase helps balance the NADPH yield from the oxidative PPP branch, supporting reductive and defense without diverting all glucose to oxidative metabolism. Transketolase operates in tandem with transaldolase to orchestrate complete flux through the non-oxidative PPP; while transaldolase transfers three-carbon units, transketolase handles two-carbon transfers, ensuring efficient interconversion of sugar phosphates and metabolic adaptability to cellular needs. This complementary action is essential for , particularly in erythrocytes, where transketolase is highly expressed and the PPP provides the primary NADPH source to maintain in its reduced form against from hemoglobin . In proliferating cells, transketolase meets elevated biosynthetic demands by prioritizing ribose-5-phosphate production, thereby sustaining synthesis and rapid . In and photosynthetic , transketolase integrates into the Calvin-Benson-Bassham cycle, reversing PPP reactions to regenerate ribulose-1,5-bisphosphate for CO₂ fixation and producing glyceraldehyde-3-phosphate as a precursor for synthesis in chloroplasts. These reversible transfers link carbon assimilation to storage, with transketolase operating near equilibrium to fine-tune based on and availability. Its activity is critical for photosynthetic efficiency, as reductions impair ribulose-1,5-bisphosphate regeneration and downstream pools, ultimately limiting plant growth and accumulation.

Isoforms and Variants

Transketolase-like protein 1 (TKTL1) is encoded by a gene located on the at locus and shares approximately 61% sequence identity with the canonical transketolase (TKT). This isoform features a 38-amino-acid deletion in its N-terminal region, which alters its and results in reduced enzymatic efficiency compared to TKT, though it retains transketolase activity in catalyzing the transfer of two-carbon units in the non-oxidative . TKTL1 is implicated in supporting anaerobic glycolysis by facilitating ribose-5-phosphate production for synthesis and NADPH generation, contributing to the Warburg effect in tumor cells where it is frequently overexpressed, such as in colon, urothelial, and gastric carcinomas. Unlike TKT, which is broadly expressed and fully reversible in its reactions, TKTL1 exhibits modified substrate specificity and lower overall activity, potentially allowing adaptation to hypoxic tumor environments. Transketolase-like protein 2 (TKTL2), encoded by a on 4q32.2, displays about 64% sequence identity to TKT and is primarily expressed in testicular tissue, where it plays a role in . This isoform is considered testis-specific and contributes to total transketolase activity in germ cells, supporting metabolic demands during sperm development, though knockout studies indicate it is not essential for . TKTL2, like TKT and TKTL1, is thiamine diphosphate-dependent but shows tissue-restricted expression and has been described in some contexts as pseudogene-like due to its intronless structure; however, it demonstrates bona fide enzymatic function in structural models. In contrast to TKTL1's tumor associations, TKTL2 expression is downregulated in certain carcinomas, highlighting isoform-specific roles. Genetic variants in the TKT gene can influence transketolase activity and are associated with metabolic disorders. For instance, several single nucleotide polymorphisms (SNPs) in TKT, such as those investigated in analyses, have been linked to altered function and increased risk of diabetic in patients, potentially by modulating flux. Rare biallelic mutations in TKT cause a recessive characterized by , developmental delay, and congenital heart defects, resulting from deficient transketolase activity and impaired thiamine-dependent . In TKTL1, certain mutations lead to isoforms with altered substrate specificity and reaction kinetics compared to wild-type TKT, which may contribute to disease susceptibility in conditions like and neurodegeneration, though these variants are less well-characterized. Both isoforms differ functionally from canonical TKT in their dependency on thiamine diphosphate, with TKTL1 showing compensatory mechanisms for its structural deletions to maintain partial activity.

Clinical Significance

Transketolase relies on (TPP), the active form of (vitamin B1), as an essential cofactor for its catalytic function in the . In , insufficient TPP availability results in reduced transketolase activity, primarily due to the accumulation of inactive apoenzyme forms in tissues such as erythrocytes. This biochemical impairment disrupts carbon flux through the non-oxidative , leading to metabolic imbalances that manifest in various clinical syndromes. The erythrocyte transketolase activity coefficient (ETKAC), calculated as the percentage increase in activity upon addition of exogenous TPP, serves as a functional ; values exceeding 15-20% typically indicate , with higher thresholds (e.g., >25%) signaling severe cases. Thiamine deficiency syndromes directly linked to transketolase dysfunction include beriberi and Wernicke-Korsakoff syndrome. Beriberi, historically associated with polished rice diets, presents in "dry" form with , , and sensory disturbances due to impaired energy metabolism in neural tissues. "Wet" beriberi involves cardiovascular complications like and from similar metabolic disruptions. Wernicke-Korsakoff syndrome, prevalent in chronic alcoholics, features acute with , ophthalmoplegia, and confusion, progressing to Korsakoff psychosis with and , often linked to brain lesions in thiamine-dependent regions. In both disorders, transketolase reactivation is observed following administration, with ETKAC assays confirming restoration of enzyme activity and supporting diagnosis. Rare genetic deficiencies in transketolase further highlight its critical role, independent of thiamine status. Transketolase deficiency (OMIM 617044) is an autosomal recessive disorder caused by biallelic mutations in the TKT gene, such as the missense variant p.Arg318Cys, which impairs enzyme stability and reduces residual activity to approximately 25%. Affected individuals exhibit , , , absent or delayed speech, and congenital heart defects like ventricular septal defects. Biochemical hallmarks include elevated polyols (e.g., , arabitol) in plasma and due to pathway backlog, underscoring the enzyme's irreplaceable function in and NADPH production. Recent advancements in the 2020s have refined ETKAC assay protocols for detecting in high-risk populations, such as (ICU) patients with , where prevalence can reach 70%. These updates emphasize standardized and cutoff interpretations to account for inflammatory confounders, enabling earlier intervention and improved outcomes in sepsis-induced metabolic stress.

Associations with Cancer and Other Diseases

Transketolase-like protein 1 (TKTL1), an isoform of transketolase, is overexpressed in a significant proportion of various tumors, including and , where it contributes to approximately 60-70% of total transketolase activity in affected cells. This overexpression promotes flux through the non-oxidative , enhancing the production of ribose-5-phosphate for nucleotide synthesis and supporting rapid tumor and survival. In , high TKTL1 expression correlates with poor disease-free survival, particularly in cases with synchronous liver metastases. Similarly, in , TKTL1 levels are elevated and associated with higher tumor grades. Recent studies have linked inhibition of transketolase activity to tumor suppression. For instance, downregulation of TKTL1 sensitizes cells to hypoxia and , reducing cell viability and migration. In models, transketolase inhibition has been shown to improve prognosis by disrupting metabolic that favors tumor growth. Recent structural studies in 2025 have modeled TKTL1's , revealing distinct differences from canonical transketolase and implications for cancer metabolism and therapeutic targeting. In , particularly , transketolase activity is reduced, leading to accumulation of harmful metabolites and increased via impaired function. This reduction exacerbates nerve damage and in peripheral tissues. Animal models of streptozotocin-induced demonstrate that supplementation restores transketolase activity, mitigates , and prevents progression of neuropathy and . Transketolase levels are diminished in neurodegenerative diseases such as and . In , amyloid-beta peptides contribute to , indirectly inhibiting transketolase and promoting beta-amyloid accumulation and cognitive decline. In , the transcription factor PARIS (ZNF746) suppresses transketolase expression, disrupting the mitochondrial and leading to dopaminergic neuron loss and energy deficits. Beyond these, transketolase has associations with infectious diseases like , where IgG antibodies against transketolase epitopes serve as a 2023 diagnostic to distinguish active from latent infection and controls with high specificity.

Diagnostic Applications

Transketolase activity serves as a functional for status through the erythrocyte transketolase activity coefficient (ETKAC) , which quantifies the percentage increase in activity upon addition of (TPP). The measures basal transketolase activity in hemolysates and compares it to stimulated activity after TPP supplementation, with the ETKAC calculated as [(stimulated activity - basal activity) / basal activity] × 100. The protocol involves spectrophotometric monitoring of NADH oxidation to NAD⁺ at 340 nm in a coupled reaction with glyceraldehyde-3-phosphate dehydrogenase and xylulose-5-phosphate as substrates, typically performed in a 96-well format for high-throughput analysis. This assay is widely applied to screen for in at-risk populations, such as chronic alcoholics and patients post-bariatric , where impairs thiamine absorption and utilization. An ETKAC value greater than 25% indicates severe , supporting the diagnosis of conditions like when combined with clinical criteria, as outlined in established laboratory guidelines. An emerging diagnostic tool involves an detecting IgG antibodies against transketolase epitopes to differentiate active from latent TB infection. Validated in 2023 on 292 subjects, the anti-TK IgG using transketolase peptide TKT3 achieved 84.5% sensitivity and 95% specificity for active TB, with significantly elevated antibody levels in active cases compared to latent infections. Despite its utility, the ETKAC assay has limitations, including false-normal results in liver disease due to reduced apotransketolase levels that mask thiamine unsaturation effects. Additionally, the assay measures total transketolase activity without distinguishing isoforms, potentially complicating interpretation in conditions with isoform-specific dysregulation.

Therapeutic Potential

Transketolase (TKT) has emerged as a promising therapeutic target in due to its central role in the non-oxidative (PPP), which supports synthesis and balance in rapidly proliferating s. Inhibitors targeting TKT, such as oxythiamine—a (TPP) analog—act as antimetabolites by competitively binding the enzyme's cofactor site, thereby disrupting PPP flux and reducing tumor cell proliferation. Preclinical studies have demonstrated that oxythiamine inhibits TKT activity, leading to altered dynamics of protein acetylation and decreased viability in cancer cell lines, particularly when combined with inhibitors to exploit metabolic vulnerabilities. The isoform transketolase-like 1 (TKTL1), often overexpressed in various cancers, presents a more specific target for isoform-selective interventions. Small interfering RNA (siRNA) mediated knockdown of TKTL1 has shown efficacy in preclinical models by impairing tumor cell rap and full viability, with studies up to 2024 highlighting its potential to suppress glycolysis and PPP activity without broadly affecting normal TKT function. Additionally, novel TKT inhibitors like oroxylin A and triazole-based TPP mimetics have been explored for their ability to elevate oxidative stress in cancer cells, enhancing sensitivity to immunotherapies and other treatments. On the activation front, high-dose supplementation (typically 100-300 mg/day) or its derivatives like enhances TKT activity by increasing cofactor availability, offering therapeutic benefits in conditions linked to TKT dysfunction, such as . Clinical trials and reviews indicate that supplementation improves neuropathic symptoms and , with reported reductions in pain scores by up to 30% in responsive patients, attributed to redirection of metabolic flux away from harmful . A 2022 supports these findings, noting consistent symptom alleviation across multiple studies, though long-term efficacy requires further validation. However, challenges persist, including achieving isoform selectivity to spare essential TKT functions in non-cancerous tissues and mitigating off-target toxicity from systemic inhibition.

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