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Structure of the cofactor-binding site in TBTKT. (a) Illustration showing TPP cofactor as cyan sticks, Mg²⁺ as a cyan sphere as well as selected amino acid residues in stick representation. Amino acids contributed by different monomers are indicated by different colour-coding. (b) Superposition of the TBTKT (green) to human TKT (silver) with cofactors TPP and Mg²⁺, and selected residues in stick representation.
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The transketolase (TKT) enzyme in Mycobacterium tuberculosis represents a novel drug target for tuberculosis treatment and has low homology with the orthologous human enzyme. Here, we report on the structural and kinetic characterization of the transketolase from M. tuberculosis (TBTKT), a homodimer whose monomers each comprise 700 amino acids. We...
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Citations
... TK was proposed as a potential target in the search of new drug against the pathogen Plasmodium falciparum (Joshi et al. 2008;Hasan et al. 2015). In Mycobacterium tuberculosis, survival and pathogenicity are dependent of arabinogalactane, a heteropolysaccharide of the cell wall, derived from D-ribose-5-phosphate synthetized in the PPP (Fullam et al. 2012). Moreover, Human TK is able to discriminate TPP from 2'methyl-thiamnie pyrophosphate while Escherichia coli TK (ecTK) is not, evidencing the difference of selectivity between both enzymes (Rabe von Pappenheim et al. 2020). ...
... Induction of the vvTK expression was done by adding IPTG (0.5 mM) and thiamine (10 μM) when the optical density at 600 nm reached a value of 0.6-0.8 (Fullam et al. 2012). Cultures were maintained at 20 C for 20 h before bacteria were harvested by centrifugation (3000 Â g) at 4 C, washed with deionized water and frozen at À80 C. Bacteria pellets were suspended in 50 mM pH 8.0 sodium phosphate supplemented by 300 mM NaCl and cells were disrupted by sonication. ...
... The two subunits have an RMSD of 1.135 Å evidencing a perfect symmetry of the protein crystal. A single vvTK chain is formed by three domains separated by two linkers (Figure 1b) (Mitschke et al. 2010;Fullam et al. 2012). The PP domain (Met 1-Leu 275) contains the interaction site with the pyrophosphate (PP) moiety of TPP. ...
Vibrio vulnificus (vv) is a multidrug‐resistant human bacterial pathogen whose prevalence is expected to increase over the years. Transketolases (TK), transferases catalyzing two reactions of the nonoxidative branch of the pentose‐phosphate pathway and therefore linked to several crucial metabolic pathways, are potential targets for new drugs against this pathogen. Here, the vvTK is crystallized and its structure is solved at 2.1 Å. A crown of 6 histidyl residues is observed in the active site and expected to participate in the thiamine pyrophosphate (cofactor) activation. Docking of fructose‐6‐phosphate and ferricyanide used in the activity assay, suggests that both substrates can bind vvTK simultaneously. This is confirmed by steady‐state kinetics showing a sequential mechanism, on the contrary to the natural transferase reaction which follows a substituted mechanism. Inhibition by the I38‐49 inhibitor (2‐(4‐ethoxyphenyl)‐1‐(pyrimidin‐2‐yl)‐1H‐pyrrolo[2,3‐b]pyridine) reveals for the first time a cooperative behavior of a TK and docking experiments suggest a previously undescribed binding site at the interface between the pyrophosphate and pyridinium domains.
... Many Bradyrhizobium isolates contain active ribulose-1,5-bisphosphate carboxylase oxygenase, which catalyzes the first stage of the Calvin-Benson-Bassham cycle (CBB) involved in carbon fixation [92]. Transketolase, an important enzyme that catalyzes the interconversion of sugars in both the CBB cycle and the pentose phosphate pathway, is also found in most Bradyrhizobium members [93,94]. The diverse metabolism in both nitrogen and carbon cycles may explain the intrinsic role of Bradyrhizobium in the determined networks. ...
Replanting is a widely used method for improving the health and carbon sequestration capacity of degraded forests. However, its impact on soil carbon pools remains controversial. This study investigated the effects of replanting broadleaf Phoebe bournei (Hemsl.) Yang in a typical degraded fir forest. Soil carbon content, nutrient levels, and microbial community structure and function were measured at 0, 5, 8, and 12 years after replanting. The degraded fir forests were originally limited in nitrogen and phosphorus. Phoebe bournei replanting significantly increased soil total carbon but reduced total nitrogen and phosphorus levels, resulting in increased soil carbon:nitrogen, carbon:phosphorus, and nitrogen:phosphorus ratios. Microbial biomass carbon, nitrogen, and phosphorus were all significantly reduced, whereas microbial carbon:phosphorus and nitrogen:phosphorus ratios were enhanced. Enzyme activities related to nutrient cycling and carbon decomposition (acidic invertase, polyphenol oxidase, peroxidase, urase, nitrate reductase, and acidic phosphatase activities) were significantly lowered by replanting. Microbial richness and diversity significantly increased, and microbial community composition changed significantly due to replanting. Structural equation modeling revealed the significant role of total phosphorus in microbial biomass, microbial community composition, and enzyme activity, highlighting it as the main factor accelerating soil carbon accumulation. Network analysis identified Leifsonia, Bradyrhizobium, and Mycolicibacterium members as key microbial players in the soil carbon cycle. In summary, P. bournei replanting exacerbated soil phosphorus deficiency, leading to a decrease in soil microbial biomass and changes in community structure, reduced nutrient cycling and carbon-decomposition-related enzyme activities, less litter decomposition, and increased organic carbon accumulation. These findings demonstrate the importance of nutrient limitation in promoting soil carbon accumulation and offer new insights for soil carbon regulation strategies in forestry.
... One of these high-performance clones had homology to the transketolase of Mycobacterium tuberculosis (M.tb) (14, 16). Transketolase (TKT) is an essential enzyme for the intracellular growth of M.tb (17), and it is the key enzyme in the non-oxidative pentose phosphate pathway (17)(18)(19). It catalyzes the reversible transfer of a two-carbon ketol group from sedoheptulose-7-phosphate to glyceraldehyde-3-phosphate, thereby producing xylulose-5-phosphate and ribose-5-phosphate. ...
... The TKT structure from most species is arranged into three domains: Domains I (1 to 322), II (323 to 527), and III (528 to 700). Domain III comprises the last approximately 170 amino acids and is involved in the regulation of the enzyme activity, and most mutations have been reported in this domain (17). M.tb TKT has 26% sequence homology with Homo sapiens and 43.8% sequence homology with S. aureus transketolase. ...
... We found that our discovered TKTm peptide (17 AA) has similarity to the M.tb transketolase on the AA sequence of THQPI, spanning from AAs 562 to 566 of M.tb TKT (Fig. 2B). It is the core domain III of M.tb TKT, which is the part of the open reading frame that contains the specific sequence motif of THDSIGLGEDGPTHQPIE [17]. Because of the detection of the abundance of the IgG antibody against the TKTm epitope in the sera of TB patients, we investigated whether designing peptides corresponding only to M.tb TKT, but not the other organisms, could improve the sensitivity and specificity of the ELISA. ...
There is an unmet need for a point-of-care, nonsputum-based TB test. Through the immunoscreening of a novel T7 phage library, we identified classifiers that specifically bind to IgGs in active TB sera.
... Structural and biochemical characterizations of TK from various organisms: bacteria (Asztalos et al. 2007;Fullam et al. 2011;Lukacik et al. 2015), yeast (Fiedler et al. 2002;Hsu et al. 2016), plant (Gerhard et al. 2003, and animal (Veitch et al. 2004;Mitschke et al. 2010), have been reported. The catalytic reaction mechanism was proposed based on high-resolution X-ray structures of reaction intermediates, in which TK forms a covalent intermediate of a substrate and TPP (Lüdtke et al. 2013;Dai et al. 2019). ...
Transketolase is a key enzyme in the pentose phosphate pathway in all organisms, recognizing sugar phosphates as substrates. Transketolase with a cofactor of thiamine pyrophosphate catalyzes the transfer of a 2-carbon unit from D-xylulose-5-phosphate to D-ribose-5-phosphate (5-carbon aldose), giving D-sedoheptulose-7-phosphate (7-carbon ketose). Transketolases can also recognize non-phosphorylated monosaccharides as substrates, and catalyze the formation of non-phosphorylated 7-carbon ketose (heptulose), which has attracted pharmaceutical attention as an inhibitor of sugar metabolism. Here, we report the structural and biochemical characterizations of transketolase from Thermus thermophilus HB8 (TtTK), a well-characterized thermophilic Gram-negative bacterium. TtTK showed marked thermostability with maximum enzyme activity at 85 °C, and efficiently catalyzed the formation of heptuloses from lithium hydroxypyruvate and four aldopentoses: D-ribose, L-lyxose, L-arabinose, and D-xylose. The X-ray structure showed that TtTK tightly forms a homodimer with more interactions between subunits compared with transketolase from other organisms, contributing to its thermal stability. A modeling study based on X-ray structures suggested that D-ribose and L-lyxose could bind to the catalytic site of TtTK to form favorable hydrogen bonds with the enzyme, explaining the high conversion rates of 41% (D-ribose) and 43% (L-lyxose) to heptulose. These results demonstrate the potential of TtTK as an enzyme producing a rare sugar of heptulose. KEY POINTS: • Transketolase catalyzes the formation of a 7-carbon sugar phosphate • Structural and biochemical characterizations of thermophilic transketolase were done • The enzyme could produce non-phosphorylated 7-carbon ketoses from sugars.
... 9,10 For these purposes, TKs from spinach, 11,12 maize, 13 Saccharomyces cerevisiae, 14−17 Escherichia coli, 18−20 and more recently from thermophilic microorganisms such as Geobacillus stearothermophilus 21,22 were characterized, in some cases improved by mutagenesis, and used in biocatalysis. TKs from Plasmodium falciparum (malaria), 23 Mycobacterium tuberculosis (tuberculosis), 24 and humans 25 were identified and studied for novel therapeutic approaches. ...
... TK enzymes are found throughout nature and have been isolated and structurally characterized from a number of different organisms including E. coli (Littlechild et al., 1995;Martin, 2008;Ludtke et al., 2013), Thermus thermophilus (PDB: 2E6K), Saccharomyces cerevisiae (Lindqvist et al., 1992;Nikkola et al., 1994), maize (Gerhardt et al., 2003), Leishmania mexicana (Veitch et al., 2004), Mycobacterium tuberculosis (Fullam et al., 2012), and human (Mitschke et al., 2010). Most TKs have a monomeric molecular mass ∼70 kDa and are active as homodimers with amino acid residues from each monomer contributing to the two active sites. ...
A novel transketolase has been reconstituted from two separate polypeptide chains encoded by a ‘split-gene’ identified in the genome of the hyperthermophilic bacterium, Carboxydothermus hydrogenoformans. The reconstituted active α2β2 tetrameric enzyme has been biochemically characterized and its activity has been determined using a range of aldehydes including glycolaldehyde, phenylacetaldehyde and cyclohexanecarboxaldehyde as the ketol acceptor and hydroxypyruvate as the donor. This reaction proceeds to near 100% completion due to the release of the product carbon dioxide and can be used for the synthesis of a range of sugars of interest to the pharmaceutical industry. This novel reconstituted transketolase is thermally stable with no loss of activity after incubation for 1 h at 70°C and is stable after 1 h incubation with 50% of the organic solvents methanol, ethanol, isopropanol, DMSO, acetonitrile and acetone. The X-ray structure of the holo reconstituted α2β2 tetrameric transketolase has been determined to 1.4 Å resolution. In addition, the structure of an inactive tetrameric β4 protein has been determined to 1.9 Å resolution. The structure of the active reconstituted α2β2 enzyme has been compared to the structures of related enzymes; the E1 component of the pyruvate dehydrogenase complex and D-xylulose-5-phosphate synthase, in an attempt to rationalize differences in structure and substrate specificity between these enzymes. This is the first example of a reconstituted ‘split-gene’ transketolase to be biochemically and structurally characterized allowing its potential for industrial biocatalysis to be evaluated.
... Transketolase (TK), also called glycolaldehyde transferase, is an enzyme that uses thiamine pyrophosphate (TPP) as its coenzyme and a divalent metal cation (Mg 2+ or Ca 2+ ) in order to perform the cleavage of carbon-carbon bonds and transfer a dihydroxyethyl unit from ketose donors to aldose acceptors (Schenk, Duggleby & Nixon, 1998;Fullam, Pojer, Bergfors, Jones & Cole, 2012). From an organic chemistry perspective, the latter seems not to be a trivial task, but a challenge for cellular chemistry due to its occurrence in an aqueous media and with phosphorylated and non-phosphorylated sugars as substrate (Miyamoto & Ohta, 2007). ...
The temporal exchange of the electrophilic/nucleophilic character of an atom by chemical manipulation is known in organic chemistry as umpolung. This inversion of polarity allows the exploration of new synthetic possibilities which cannot be achieved from the normal reactivity of functional groups. It is not only a useful synthetic tool, but it is also utilized by certain enzymes during biochemical reactions in living organisms. Thiamine pyrophosphate dependent enzymes, such as pyruvate decarboxylase, pyruvate dehy-drogenase, α-ketoglutarate dehydrogenase, branched-chain α-keto acids dehydrogenase and transketolase, provide clear examples of umpolung in cellular reactions that fulfill different purposes. In this review, after a discussion regarding the meaning of the term umpolung found in the chemical literature, the reaction mechanisms and the biochemical meaning of transformations carried out by the aforemen-tioned enzymes are analyzed.
... Transketolase (TKT) is an enzyme of the nonoxidative branch of the PPP involved in two main reversible enzymatic reactions: i) Fructose-6-P + Glyceraldehyde-3-P <-> Erythrose-4-P + Xylulose-5-P; and ii) Sedoheptulose-7-P + Glyceraldehyde-3-P <-> Ribose-5-P + Xylulose-5-P (Supplementary Figure S2A). Several TKT structures have been solved [35] and allow the modelisation of the S. aureus TKT (Figure 1A). In contrast, the ∆tkt mutant strain was unable to grow in chemically defined medium [31] supplemented with ribose (which can be converted to Ribose-5-phospate by ribokinase) (Supplementary Figure S2C). ...
... TKT inhibitors are currently actively tested in cancer therapy [47] and TKT could constitute an efficient target against tuberculosis [35] and malaria [48]. The present work, highlighting an unprecedentedly reported role of transketolase in S. aureus intracellular survival, suggests that modulation of the pentose phosphate pathway activity may also represent an interesting mean to fight S. aureus infections. ...
Staphylococcus aureus is a leading cause of both acute and chronic infections in humans. The importance of the pentose phosphate pathway (PPP) during S. aureus infection is currently largely unexplored. Here, we focused on one key PPP enzyme, transketolase. We showed that inactivation of the unique gene encoding transketolase activity in S. aureus USA300 (∆tkt) led to drastic metabolomic changes. Using time-lapse video imaging and mice infection, we observed a major defect of the ∆tkt strain compared to wild-type strain in early intracellular proliferation and in the ability to colonise kidneys. Transcriptional activity of the two master regulators Sigma B and RpiRc was drastically reduced in the ∆tkt mutant during host cells invasion. The concomitant increased RNAIII transcription, suggests that TKT -or a functional PPP- strongly influences the ability of S. aureus to proliferate within host cells by modulating key transcriptional regulators.
... Transketolase is a homodimeric enzyme that catalyses the reversible transfer of two carbons from a ketose donor substrate to an aldose acceptor substrate (Zhao and Zhong, 2009;Fullam et al., 2012). TK is the most active enzyme involved in the non-oxidative branch of the pentose phosphate pathway and is responsible for generating the ribose-5-P molecules necessary for nucleic acid synthesis (Schaaffgerstenschlager and Zimmermann, 1993) (Kim et al., 2012). ...
Glycated haemoglobin (HbA1c) is the most important marker of hyperglycaemia in diabetes mellitus. We show that D-ribose reacts with haemoglobin, thus yielding HbA1c. Using mass spectrometry, we detected glycation of haemoglobin with D-ribose produces 10 carboxylmethyllysines (CMLs). The first-order rate constant of fructosamine formation for D-ribose was approximately 60 times higher than that for D-glucose at the initial stage. Zucker Diabetic Fatty (ZDF) rat, a common model for type 2 diabetes mellitus (T2DM), had high levels of D-ribose and HbA1c, accompanied by a decrease of transketolase (TK) in the liver. The administration of benfotiamine, an activator of TK, significantly decreased D-ribose followed by a decline in HbA1c. In clinical investigation, T2DM patients with high HbA1c had a high level of urine D-ribose, though the level of their urine D-glucose was low. That is, D-ribose contributes to HbA1c, which prompts future studies to further explore whether D-ribose plays a role in the pathophysiological mechanism of T2DM.
... For example, the detailed description of the activation of cofactor ThDP is still absent. In the previous studies [19][20][21][22][23][24][25] it is assumed that the conserved glutamate residue is necessary for the activation of ThDP. However, the proton exchange at the C2-carbon of thiazolium group can still occur without the assistance of the glutamate residue. ...
Glyoxylate carboligase (GCL) catalyzes the ligation of two molecules of glyoxylate to form tartronate semialdehyde (TSA) and carbon dioxide. GCL is unique among ThDP-dependent enzymes because it lacks the canonical glutamate in the active site which is thought to be highly necessary for other ThDP enzymes. In this paper, the catalytic reactions of GCL and its mutant V51E have been explored using a combined QM/MM approach. On the basis of our calculations, the following important points have been obtained: (1) although the glutamate residue is absent in the active site of GCL, the activation of cofactor ThDP is still quite easy with a very low energy barrier of 5.0 kcal/mol; (2) the catalytic cycle can still proceed in the case where no other potential acid–base side chain exists in the active site; (3) for the wild-type GCL, the energy barrier of the most energy-requiring step is 17.9 kcal/mol, which agrees well with the experimental observations; (4) for V51E mutant, the concerted formation of the product TSA and regeneration of the ThDP ylide is calculated to be the rate-limiting step with an energy barrier of 19.5 kcal/mol. It is slightly higher than that of the wild-type GCL (19.5 vs. 17.9 kcal/mol), which can well explain the relatively lower activity of the V51E mutant than the wild-type enzyme. Our results may provide help for further understanding the catalytic mechanism of GCL.
