Transketolase Activity Research Articles

Metabolic evolution of two reducing equivalent-conserving pathways for high-yield succinate production in Escherichia coli

Reducing equivalents are an important cofactor for efficient synthesis of target products. During metabolic evolution to improve succinate production in Escherichia coli strains, two reducing equivalent-conserving pathways were activated to increase succinate yield. The sensitivity of pyruvate dehydrogenase to NADH inhibition was eliminated by three nucleotide mutations in the lpdA gene. Pyruvate dehydrogenase activity increased under anaerobic conditions, which provided additional NADH. The pentose phosphate pathway and transhydrogenase were activated by increased activities of transketolase and soluble transhydrogenase SthA. These data suggest that more carbon flux went through the pentose phosphate pathway, thus leading to production of more reducing equivalent in the form of NADPH, which was then converted to NADH through soluble transhydrogenase for succinate production. Reverse metabolic engineering was further performed in a parent strain, which was not metabolically evolved, to verify the effects of activating these two reducing equivalent-conserving pathways for improving succinate yield. Activating pyruvate dehydrogenase increased succinate yield from 1.12 to 1.31mol/mol, whereas activating the pentose phosphate pathway and transhydrogenase increased succinate yield from 1.12 to 1.33mol/mol. Activating these two pathways in combination led to a succinate yield of 1.5mol/mol (88% of theoretical maximum), suggesting that they exhibited a synergistic effect for improving succinate yield.

The detrimental effects of acute hyperglycemia on myocardial glucose uptake

AimsAlthough acute hyperglycemic (AHG) episodes are linked to lower glucose uptake, underlying mechanisms remain unclear. We hypothesized that AHG triggers reactive oxygen species (ROS) production and increases non-oxidative glucose pathway (NOGP) activation, i.e. stimulation of advanced glycation end products (AGE), polyol pathway (PP), hexosamine biosynthetic pathway (HBP), PKC; thereby decreasing cardiac glucose uptake. Main methodsH9c2 cardiomyoblasts were exposed to 25mM glucose for 24h vs. 5.5mM controls±modulating agents during the last hour of glucose exposure: a) antioxidant #1 for mitochondrial ROS (250μM 4-OHCA), b) antioxidant #2 for NADPH oxidase-generated ROS (100μM DPI), c) NOGP inhibitors — 100μM aminoguanidine (AGE), 5μM chelerythrine (PKC); 40μM DON (HBP); and 10μM zopolrestat (PP). ROS levels (mitochondrial, intracellular) and glucose uptake were evaluated by flow cytometry. Key findingsAHG elevated ROS, activated NOGPs and blunted glucose uptake. Transketolase activity (pentose phosphate pathway [PPP] marker) did not change. Respective 4-OHCA and DPI treatment blunted ROS production, diminished NOGP activation and normalized glucose uptake. NOGP inhibitory studies identified PKCβII as a key downstream player in lowering insulin-mediated glucose uptake. When we employed an agent (benfotiamine) known to shunt flux away from NOGPs (into PPP), it decreased ROS generation and NOGP activation, and restored glucose uptake under AHG conditions. SignificanceThis study demonstrates that AHG elicits maladaptive events that function in tandem to reduce glucose uptake, and that antioxidant treatment and/or attenuation of NOGP activation (PKC, polyol pathway) may limit the onset of insulin resistance.

B vitamins reduce body weight gain and increase metabolism related enzyme activities in rats (828.11)

B vitamins as enzyme cofactors play an important role in energy metabolism. The aim of this study was to determine whether B vitamins can affect body weight (BW) and activity of energy metabolism‐related enzymes in an animal model. Fifty Wistar rats were randomly divided to 3 groups and were fed a diet containing 10% fat (LF), a diet containing 45% fat (HF), or a HF diet supplemented with B vitamins (100 mg/kg BW for both thiamine and riboflavin, 250 mg/kg niacin) (HF+B). After rats were fed for 12 weeks, HF group had significantly larger weight gain than LF group (285.2±14.8 g versus 159.1±53.0 g, P<0.05), while HF+B group had a weight gain (154.5±58.4 g) not significantly different from that in LF group. In addition, compared to HF group, HF+B group had significantly lower levels of plasma total cholesterol (1.19±0.18 mM versus 1.59±0.30 mM), and triglyceride (0.76±0.34 mM versus 1.55±0.40 mM). Activity of transketolase, glutathione reductase, and Na+/K+ adenosine triphosphatase was higher in HF+B group than that in HF group. In conclusion, B vitamin supplementation reduces HF‐induced weigh gain and increase in blood lipids, which may be associated with enhanced activity of the energy metabolism‐related enzymes.Grant Funding Source: NSFC‐81172662

Characterization of two transketolases encoded on the chromosome and the plasmid pBM19 of the facultative ribulose monophosphate cycle methylotroph Bacillus methanolicus

BackgroundTransketolase (TKT) is a key enzyme of the pentose phosphate pathway (PPP), the Calvin cycle and the ribulose monophosphate (RuMP) cycle. Bacillus methanolicus is a facultative RuMP pathway methylotroph. B. methanolicus MGA3 harbors two genes putatively coding for TKTs; one located on the chromosome (tkt C ) and one located on the natural occurring plasmid pBM19 (tkt P ).ResultsBoth enzymes were produced in recombinant Escherichia coli, purified and shown to share similar biochemical parameters in vitro. They were found to be active as homotetramers and require thiamine pyrophosphate for catalytic activity. The inactive apoform of the TKTs, yielded by dialysis against buffer containing 10 mM EDTA, could be reconstituted most efficiently with Mn2+ and Mg2+. Both TKTs were thermo stable at physiological temperature (up to 65°C) with the highest activity at neutral pH. Ni2+, ATP and ADP significantly inhibited activity of both TKTs. Unlike the recently characterized RuMP pathway enzymes fructose 1,6-bisphosphate aldolase (FBA) and fructose 1,6-bisphosphatase/sedoheptulose 1,7-bisphosphatase (FBPase/SBPase) from B. methanolicus MGA3, both TKTs exhibited similar kinetic parameters although they only share 76% identical amino acids. The kinetic parameters were determined for the reaction with the substrates xylulose 5-phosphate (TKTC: kcat/KM: 264 s-1 mM-1; TKTP: kcat/KM: 231 s-1 mM) and ribulose 5-phosphate (TKTC: kcat/KM: 109 s-1 mM; TKTP: kcat/KM: 84 s-1 mM) as well as for the reaction with the substrates glyceraldehyde 3-phosphate (TKTC: kcat/KM: 108 s-1 mM; TKTP: kcat/KM: 71 s-1 mM) and fructose 6-phosphate (TKTC kcat/KM: 115 s-1 mM; TKTP: kcat/KM: 448 s-1 mM).ConclusionsBased on the kinetic parameters no major TKT of B. methanolicus could be determined. Increased expression of tkt P , but not of tkt C during growth with methanol [J Bacteriol 188:3063–3072, 2006] argues for TKTP being the major TKT relevant in the RuMP pathway. Neither TKT exhibited activity as dihydroxyacetone synthase, as found in methylotrophic yeast, or as the evolutionary related 1-deoxyxylulose-5-phosphate synthase. The biological significance of the two TKTs for B. methanolicus methylotrophy is discussed.

Downregulation of transketolase activity is related to inhibition of hippocampal progenitor cell proliferation induced by thiamine deficiency.

In animal experiments, hippocampal neurogenesis and the activity of thiamine-dependent transketolase decrease markedly under conditions of thiamine deficiency. To further investigate the effect of thiamine deficiency on the proliferation of hippocampal progenitor cells (HPCs) and the potential mechanisms involved in this effect, we cultured HPCs in vitro in the absence of thiamine and found that proliferation and transketolase activity were both significantly repressed. Furthermore, specific inhibition of transketolase activity by oxythiamine strongly inhibited HPC proliferation in a dose-dependent manner. However, thiamine deficiency itself inhibited the proliferation to a greater degree than did oxythiamine. Taken together, our results suggest that modulation of transketolase activity might be one of the mechanisms by which thiamine regulates the proliferation of hippocampal progenitor cells.

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Introduction: Type B lactic acidosis is a high-anion gap metabolic acidosis that occurs in the absence of tissue hypoxia. It involves the underutilization of lactate or compromised lactate metabolism. Liver disease, pyruvate dehydrogenase (thiamine) deficiency, inhibition of gluconeogenesis, and uncoupling of oxidative phosphorylation are the most common causes. We present a 4-year-old Caucasian male with medulloblastoma s/p resection undergoing treatment with Vincristine, proton beam therapy and steroids. He presented to the PICU with presumed septic shock that was treated with fluids, vasopressors, antibiotics and stress dose steroids. He was neutropenic and exhibited low-grade DIC. He demonstrated high-output renal failure with electrolyte wasting despite treatment. His mentation was impaired. His initial pH was 7.25 and his lactate ranged between10-12 mEq/L. An abdominal radiograph on admission was suggestive of pneumatosis. A subsequent CT scan of the abdomen did not reveal significant bowel inflammation or perforation. The patient improved and vasopressor support was stopped after 48 hours, yet his lactic acidosis persisted. Lactate levels peaked hospital day 3 at 19 mEq/L in the absence of hypoperfusion and was accompanied by profound hyperglycemia. A concomitant abdominal radiograph was suspicious for free air so the patient underwent exploratory laparoscopy that did not show evidence of ischemia or perforation. Because a type B lactic acidosis was suspected, intravenous thiamine and L-carnitine were given empirically and the lactate normalized over several hours, as did his urine output, and his mentation. Thiamine activity levels (transketolase activity in red blood cells) were not determined pre-therapy. The lactate/pyruvate ratio decreased to 10 from 20 after treatment. His risk factors for thiamine deficiency included malignancy, intractable emesis, and need for chronic total parenteral nutrition (TPN), which lacked MVI for several weeks due to national shortage. Thiamine is a cofactor for pyruvate dehydrogenase, which is a key enzyme in the anaerobic oxidation of glucose. The most dangerous sequela of thiamine deficiency is the CNS injury (Wernicke-Korsakoff syndrome). This patient recovered fully, finished his radiation therapy during his hospital course and was discharged. Patients receiving TPN without MVI supplementation are at risk for thiamine deficiency and its associated life-threatening complications.

The effect of magnesium administration on erythrocyte transketolase activity in alcoholic patients treated with thiamine

Chronic alcoholic patients are at increased risk of developing deficiencies of thiamine and magnesium. Thiamine is an essential co-factor for a number of enzymes involved in carbohydrate metabolism and requires optimal levels of magnesium for biological function. However, whilst thiamine supplementation is well established for the treatment of alcoholic patients, the importance of magnesium is often overlooked. We describe the effect of concurrent thiamine and magnesium administration on the activity of the thiamine-dependent enzyme erythrocyte transketolase in a cohort of chronic alcoholic patients. Baseline erythrocyte transketolase activities were measured on blood samples collected from 36 chronic alcoholic patients presenting acutely to the Accident and Emergency department. Patients received either intravenous Pabrinex (thiamine) supplemented with magnesium sulphate (n = 18) or Pabrinex only (n = 18). Post-treatment bloods were collected for re-assessment of erythrocyte transketolase activity. The change in transketolase activities (pre-vs. post-treatment) between the two patient groups were compared by Mann-Whitney U test. The increase in transketolase activity following treatment in the cohort receiving Pabrinex supplemented with magnesium sulphate was significantly greater (p = 0.018) than that produced in the cohort receiving Pabrinex alone. In the acute management of a sample of chronic alcoholic patients, those receiving magnesium sulphate with Pabrinex have higher increases in erythrocyte transketolase activity compared with those receiving Pabrinex alone. We conclude that concurrent magnesium administration with Pabrinex may be required for enabling full efficacy of Pabrinex treatment, as demonstrated by its positive effect on erythrocyte transketolase activity.

Current Options and Perspectives in the Treatment of Diabetic Neuropathy

We aimed to summarise recent advances in the therapy of diabetic neuropathy. Although all therapeutic choices in the treatment of diabetes mellitus itself are based on clear pathophysiological basis, this approach is less present in the treatment of the "forgotten complication", diabetic neuropathy. As part of pathogenetic oriented treatment, the role of glycemic control and cardiovascular risk factors are reviewed. The mode of action of benfotiamine is based on inhibition of key alternative pathways, including the polyol, hexosamine, protein-kinase-C pathways, and inhibition of advanced glycation end products formation, just as on activation of transketolase. Alpha- lipoic-acid is considered as the most potent antioxidant. Other forms of pathogenetic oriented treatment, including actovegin, will be summarised. The anticonvulsants gabapentin and pregabalin, as well as the antidepressant duloxetine represent the most important new drugs among agents for symptomatic relief. Most likely, we should offer combination treatment to our patients much more often, first of all combination of pathogenetic and symptomatic drugs. Finally, the broad spectrum of non-pharmacological treatment will be reviewed.

Low iron availability and phenolic metabolism in a wild plant species (Parietaria judaica L.)

Plant phenolics encompass a wide range of aromatic compounds and functions mainly related to abiotic and biotic environmental responses. In calcareous soils, the presence of bicarbonate and a high pH cause a decrease in iron (Fe) bioavailability leading to crop yield losses both qualitatively and quantitatively. High increases in phenolics were reported in roots and root exudates as a consequence of decreased Fe bioavailability suggesting their role in chelation and reduction of inorganic Fe(III) contributing to the mobilization of Fe oxides in soil and plant apoplast. Shikimate pathway represents the main pathway to provide aromatic precursors for the synthesis of phenylpropanoids and constitutes a link between primary and secondary metabolism. Thus the increased level of phenolics suggests a metabolic shift of carbon skeletons from primary to secondary metabolism. Parietaria judaica, a spontaneous plant well adapted to calcareous environments, demonstrates a high metabolic flexibility in response to Fe starvation. Plants grown under low Fe availability conditions showed a strong accumulation of phenolics in roots as well as an improved secretion of root exudates. P. judaica exhibits enhanced enzymatic activities of the shikimate pathway. Furthermore, the non-oxidative pentose phosphate pathway, through the transketolase activity supplies erythrose-4-phosphate, is strongly activated. These data may indicate a metabolic rearrangement modifying the allocation of carbon skeletons between primary and secondary metabolism and the activation of a nonoxidative way to overcome a mitochondrial impairment. We suggest that high content of phenolics in P. judaica play a crucial role in its adaptive strategy to cope with low Fe availability.

Epicatechin Gallate Impairs Colon Cancer Cell Metabolic Productivity

Green tea and grape phenolics inhibit cancer growth and modulate cellular metabolism. Targeting the tumor metabolic profile is a novel therapeutic approach to inhibit cancer cell proliferation. Therefore, we treated human colon adenocarcinoma HT29 cells with the phenolic compound epicatechin gallate (ECG), one of the main catechins in green tea and the most important catechin in grape extracts, and evaluated its antiproliferation effects. ECG reduced tumor viability and induced apoptosis, necrosis, and S phase arrest in HT29 cells. Later, biochemical determinations combined with mass isotopomer distribution analysis using [1,2-(13)C2]-D-glucose as a tracer were used to characterize the metabolic network of HT29 cells in response to different concentrations of ECG. Glucose consumption was importantly decreased after ECG treatment. Moreover, metabolization of [1,2-(13)C2]-D-glucose indicated that the de novo synthesis of fatty acids and the pentose phosphate pathway were reduced in ECG-treated cells. Interestingly, ECG inhibited the activity of transketolase and glucose-6-phosphate dehydrogenase, the key enzymes of the pentose phosphate pathway. Our data point to ECG as a promising chemotherapeutic agent for the treatment of colon cancer.

Exploring mechanisms that attenuate myocardial glucose uptake in response to acute hyperglycemia: identification of a vicious metabolic cycle

Acute hyperglycemia (AHG) e.g. high post‐prandial glycemic excursions are linked to lower glucose uptake. Chronic hyperglycemia can trigger reactive oxygen species (ROS) production and increase non‐oxidative glucose pathway (NOGP) flux, i.e. polyol pathway (PP), hexosamine biosynthetic pathway (HBP), advanced glycation end‐products (AGE), PKC, and contribute to insulin resistance. Since it is unclear whether a similar scenario exists with AHG, we hypothesized that ROS plays a pivotal role in AHG‐mediated NOGP activation and decreased myocardial glucose uptake. H9c2 cardiomyoblasts were exposed to 25 mM glucose for 24 h vs. 5 mM glucose controls ± therapeutic agents during last h of glucose exposure: a) antioxidant (250 μM 4‐OHCA), b) NOGP inhibitors − 100 μM aminoguanidine (AGE); 5 μM chelerythrine (PKC); 40 μM DON (HBP); and 10 μM zopolrestat (PP). ROS levels (mitochondrial, intracellular) and glucose uptake were evaluated by flow cytometry. AHG significantly increased mitochondrial and intracellular ROS levels, activated NOGP and blunted glucose uptake. However, transketolase (TK) activity (marker of pentose phosphate pathway) was not significantly altered. By contrast, 4‐OHCA treatment significantly reduced ROS, TK activity, NOGP activation and normalized glucose uptake. Each individual NOGP inhibitor blunted activation of the other pathways examined here. Our study reveals a convergence of downstream NOGP effects and identifies a vicious metabolic cycle, i.e. AHG stimulates NOGP that further fuel its own activation by generating more oxidative stress and further diminishing glucose uptake. Antioxidant treatment may be a useful approach to counter the potential detrimental effects of pronounced, acute hyperglycemic episodes.

Are brain and heart tissue prone to the development of thiamine deficiency?

Thiamine deficiency is a continuing problem leading to beriberi and Wernicke's encephalopathy. The symptoms of thiamine deficiency develop in the heart, brain and neuronal tissue. Yet, it is unclear how rapid thiamine deficiency develops and which organs are prone to development of thiamine deficiency. We investigated these issues in a thiamine deficient animal model. Twenty-four male Lewis rats were fed a thiamine deficient diet, which contained 0.04% of normal thiamine intake. Six control rats were fed 200 μg of thiamine per day. Every week a group of six rats on the thiamine-deficient diet was sacrificed and blood, urine and tissue were stored. Blood and tissue transketolase activity, thiamine and thiamine metabolites were measured and PCR of thiamine transporter-1 (ThTr-1) was performed. Transketolase activity was significantly reduced in red blood cells, liver, lung, kidney and spleen tissue after two weeks of thiamine deficient diet. In brain tissue, transketolase activity was not reduced after up to four weeks of thiamine deficient diet. The amount of thiamine pyrophosphate was also significantly conserved in brain and heart tissue (decrease of 31% and 28% respectively), compared to other tissues (decrease of ∼70%) after four weeks of thiamine deficient diet. There was no difference between tissues in ThTr-1 expression after four weeks of thiamine deficient diet. Despite the fact that the heart and the brain are predilection sites for complications from thiamine deficiency, these tissues are protected against thiamine deficiency. Other organs could be suffering from thiamine deficiency without resulting in clinical signs of classic thiamine deficiency in beriberi and Wernicke's encephalopathy.

Synthesis of Specially Designed Probes to Broaden Transketolase Scope

AbstractEfficient, biocompatible, stereospecific strategies were developed to prepare eight probes to assay transketolase (TK) variants with new substrate specificities. The structure of these probes combines a sugar moiety (D‐threo or L‐erythro ketose, or D‐threo aldose) with the side chain of an amino acid (Ala, Leu, Val, Met, Thr) for in vivo detection of new TK activities using amino acid auxotrophs. To obtain D‐threo ketose probes, biocatalysts, such as transketolase and fructose‐6‐phosphate aldolase Ala129Ser, were used whereas L‐erythro ketoses and D‐threo aldose probes were synthesized by the way of organocatalysis or Sharpless dihydroxylation as sustainable alternative key steps to biocatalysis.

Original article Erythrocyte transketolase activity in patients with diabetic and alcoholic neuropathies

The activity of erythrocyte transketolase induced by thiamine pyrophosphate and normalized to age of the patient is a marker of thiamine metabolism disturbances with pathological consequences in the central and peripheral nervous system. The measurement of erythrocyte transketolase activity enables evaluation of the thiamine status and therapeutic decisions in the disorders of the nervous system related to its deficiency. The aim of the study was to compare different modes of expression of transketolase activity in the most frequent acquired neuropathies. The study included 29 patients with type 2 diabetes mellitus (21 males, 8 females) aged 48.3 years (range: 20-70 years) and 31 subjects with a history of alcohol dependence (23 males, 8 females) aged 54.5 years (range: 28-60 years). All participants of the study showed signs and symptoms of neuropathy. The cases in both groups presented the involvement of either axon, myelin, or both, what was evidenced by electrophysiological tests (electromyography and estimation of nerve conduction velocity). The control group consisted of 20 healthy persons aged 49.1 years (range: 23-70 years). Transketolase activity in erythrocytes (TK) was assessed by means of the spectrophotometric method. Basic TK activity was expressed as units per gram of haemoglobin (g Hb), moreover the normalized transketolase activity ratio (NTKZ) and percentage of activation of thiamine pyrophosphate (TPP) were calculated. The basal TK activity was decreased in cases with diabetic neuropathy (0.64 ± 0.342 U/g Hb, p = 0.0131), whereas in patients with alcoholic neuropathy only a trend (p = 0.058) of the decrease was noticed (0.85 ± 0.484 U/g Hb), in relation to the control group (1.005 ± 0.390 U/g Hb). Normalized transketolase activity ratio in erythrocytes has not shown any statistically significant differences. The median TPP did not indicate any thiamine deficiency, both in the group of diabetic and alcoholic neuropathy. The decrease in transketolase activity in diabetic neuropathy may be independent of thiamine deficiency. Abnormalities in transketolase activity, when expressed in three modalities: basal activity, normalized transketolase activity ratio and the activity after the stimulation with thiamine pyrophosphate may be differentiated as thiamine-dependent or resulting from posttranslational modification.

Thiamin Status and Supplementation in the Management of Diabetes Mellitus and its Vascular Comorbidities

The water soluble B vitamin, thiamin, in its coenzyme form as thiamin pyrophosphate (TPP), is necessary for key reactions in glucose metabolism. For this reason, associations between thiamin status or thiamin supplementation and diabetes have been the focus of recent research. Currently, it is not clear how occurrence of diabetes relates to parameters of thiamin status, such as plasma thiamin levels. However, there is strong evidence that the diabetic state increases urinary thiamin excretion and decreases the activity of transketolase (TK), a TPP-dependent enzyme of the hexose monophosphate (HMP) shunt in various body tissues. Impairment of TK activity and subsequent downregulation of the HMP shunt activates several pathways that contribute to vascular damage and development of diabetes-related comorbidities such as retinopathy, cardiomyopathy, and nephropathy. Thiamin supplementation has been shown to be effective in restoring TK activity in animal models of diabetes, and in type 1 and type 2 diabetic individuals. Thiamin supplementation has also been shown to be effective in preventing or reversing, either partially or completely, hyperglycemia-induced damage to vascular endothelial cells, and microalbuminuria associated with diabetic nephropathy. Here, we review from current literature examining the relationship between diabetes and thiamin status as well as intervention trials evaluating the effect of thiamin supplementation on glycemic control and prevention of vascular comorbidities of diabetes.

Transketolase -like protein 1 cannot exert transketolase activity

Transketolase -like protein 1 cannot exert transketolase activity

Is transketolase-like protein, TKTL1, transketolase?

Until recently it was assumed that the transketolase-like protein (TKTL1) detected in the tumor tissue, is catalytically active mutant form of human transketolase (hTKT). Human TKT shares 61% sequence identity with TKTL1. And the two proteins are 77% homologous at the amino acid level. The major difference is the absence of 38 amino acid residues in the N-terminal region of TKTL1. Site-specific mutagenesis was used for modifying hTKT gene; the resulting construct had a 114-bp deletion corresponding to a deletion of 38 amino acid residues in hTKT protein. Wild type hTKT and mutant variant (DhTKT) were expressed in Escherichia coli and isolated using Ni-agarose affinity chromatography. We have demonstrated here that DhTKT is devoid of transketolase activity and lacks bound thiamine diphosphate (ThDP). In view of these results, it is unlikely that TKTL1 may be a ThDP-dependent protein capable of catalyzing the transketolase reaction, as hypothesized previously.

A Δ38 Deletion Variant of Human Transketolase as a Model of Transketolase-Like Protein 1 Exhibits No Enzymatic Activity

Besides transketolase (TKT), a thiamin-dependent enzyme of the pentose phosphate pathway, the human genome encodes for two closely related transketolase-like proteins, which share a high sequence identity with TKT. Transketolase-like protein 1 (TKTL1) has been implicated in cancerogenesis as its cellular expression levels were reported to directly correlate with invasion efficiency of cancer cells and patient mortality. It has been proposed that TKTL1 exerts its function by catalyzing an unusual enzymatic reaction, a hypothesis that has been the subject of recent controversy. The most striking difference between TKTL1 and TKT is a deletion of 38 consecutive amino acids in the N-terminal domain of the former, which constitute part of the active site in authentic TKT. Our structural and sequence analysis suggested that TKTL1 might not possess transketolase activity. In order to test this hypothesis in the absence of a recombinant expression system for TKTL1 and resilient data on its biochemical properties, we have engineered and biochemically characterized a “pseudo-TKTL1” Δ38 deletion variant of human TKT (TKTΔ38) as a viable model of TKTL1. Although the isolated protein is properly folded under in vitro conditions, both thermal stability as well as stability of the TKT-specific homodimeric assembly are markedly reduced. Circular dichroism and NMR spectroscopic analysis further indicates that TKTΔ38 is unable to bind the thiamin cofactor in a specific manner, even at superphysiological concentrations. No transketolase activity of TKTΔ38 can be detected for conversion of physiological sugar substrates thus arguing against an intrinsically encoded enzymatic function of TKTL1 in tumor cell metabolism.

A pH‐Based High‐Throughput Assay for Transketolase: Fingerprinting of Substrate Tolerance and Quantitative Kinetics

A pH-based high-throughput assay method has been developed for the rapid and reliable measurement of transketolase (TK) activity. The method is based on the decarboxylation of lithium hydroxypyruvate (HPA) as a hydroxyacetyl donor with an aldehyde acceptor, using phenol red as the pH indicator. Upon release of carbon dioxide from HPA, the pH increase in the reaction mixture can be determined photometrically by the color change of the pH indicator. At low buffer concentration (2 mM triethanolamine, pH 7.5), the method is highly sensitive and allows continuous monitoring, for quantitative determination of the kinetic parameters. By using this method, the substrate specificities of the TK enzymes from Escherichia coli and Saccharomyces cerevisiae, as well as two active-site-modified variants of the E. coli TK (D469E, H26Y) were evaluated against a panel of substrate analogues; specific activities and kinetic constants could be rapidly determined. Substrate quality indicated by assay determination was substantiated with novel TK applications by using achiral 3-hydroxypropanal and 4-hydroxybutanal for preparative synthesis of chiral deoxyketose-type products. Determination of ee for the latter could be performed by chiral GC analysis, with an unambiguous correlation of the absolute configuration from rotation data. This pH-based assay method is broadly applicable and allows rapid, sensitive, and reliable screening of the substrate tolerance of known TK enzymes and variants obtained from directed evolution.

Co-occurrence of Anemia, Marginal Vitamin B6, and Folate Status and Depressive Symptoms in Older Adults

Although nutrient deficiencies are thought to play roles in the development of depression, observational studies have yielded inconsistent results. This study aimed to investigate whether multiple marginal nutrient deficiencies are associated with symptoms of depression in community-dwelling older Taiwanese. Data from 1371 elderly adults recruited from the Elderly Nutrition and Health Survey in Taiwan was used in this study. Depressive symptom scores on depressed mood and emotions affecting daily life were derived from the Medical Outcomes Study Short Form-36 (SF-36). Hemoglobin, serum ferritin, plasma vitamins B(6), B(12), and folate concentration, and erythrocyte transketolase and glutathione reductase activation coefficients were measured. After adjusting for age, gender, cognitive function, physical activity, disease history, and medication in the multivariate analysis, anemia, and marginal B(6) deficiency were significantly associated with the presence of depression symptoms, respectively. In addition, co-occurrence of vitamin B(6) with low folate level and co-occurrence of anemia either with low vitamin B(6) or with folate level were all associated with the depressive mood and with depressive emotions defined by SF-36 (odds ratios [OR] in the range of 2.32-7.13, all P values ≤.05). The magnitude of the ORs is larger when the number of deficiencies increased. Elderly people with coexisting marginal deficiencies of nutrients involved in the S-adenosylmethionine and hemoglobin production were more likely to experience depressed mood and emotion that affect daily activity. Examining status of these nutrients is worthy of consideration for older adults with depressed symptoms.