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Abstract

In plants, metabolic homeostasis—the driving force of growth and development—is achieved through the dynamic behavior of a network of enzymes, many of which depend on coenzymes for activity. The circadian clock is established to influence coordination of supply and demand of metabolites. Metabolic oscillations independent of the circadian clock, particularly at the subcellular level is unexplored. Here, we reveal a metabolic rhythm of the essential coenzyme thiamine diphosphate (TDP) in the Arabidopsis nucleus. We show there is temporal separation of the clock control of cellular biosynthesis and transport of TDP at the transcriptional level. Taking advantage of the sole reported riboswitch metabolite sensor in plants, we show that TDP oscillates in the nucleus. This oscillation is a function of a light-dark cycle and is independent of circadian clock control. The findings are important to understand plant fitness in terms of metabolite rhythms.

Introduction

Here, in addition to demonstrating the importance of TDP for central metabolism and thereby growth and development, we report on components of the intracellular transport of TDP and/or its precursors in the plant model Arabidopsis. We show that both TDP biosynthesis and transport are transcriptionally regulated by the circadian clock. Importantly, biosynthesis is temporally separated from transport and is likely an important parameter for coordination of TDP supply and demand. We have used the alternative splicing of the THIC riboswitch as a natural sensing tool to report on daily modulation of TDP levels. Indeed, levels of TDP oscillate at the intracellular level (i.e. the nucleus) peaking in abundance in the first half of the day. This peak in abundance is derivative of light-dark cycles that is independent of the circadian clock regulation.

TDP is essential for central metabolism

During seed development/maturation in plants, the nonphosphorylated B1 vitamer thiamine is stored, and upon germination is converted to TDP (Fig. ), furnishing enzymes of central metabolism in the cotyledons. Beyond this however, the development of true leaves requires biosynthesis de novo of TDP. This is corroborated by the fact that thiamine biosynthesis de novo mutants, such as thiC, cannot proceed beyond the cotyledon stage of development. The necessity of having biosynthesis de novo can be bypassed by supplementation with thiamine. Bypassing biosynthesis de novo in these previous experiments was performed by supplementing with excessive levels of thiamine (up to 100 μM). In order to test whether core metabolism is indeed dependent on TDP levels, we grew the thiC mutant in the presence of a range of thiamine concentrations (0−1.5 μM), spanning physiologically measured levels. Supplementation with thiamine had little effect on wild-type growth (Fig. ). However, the growth of thiC beyond the cotyledon stage was dependent on the level of thiamine supplementation (Fig. ). Moreover, TDP levels in these seedlings showed a clear correlation with thiamine supplementation levels (Fig. ). Furthermore, we utilized a nontargeted metabolomics approach to assess metabolite changes as a function of the thiamine supplementation level. Mapping the set of detectable metabolites onto a scheme of central metabolism illustrates the panorama of metabolite changes with and without thiamine supplementation in thiC (Supplementary Fig. ). Such changes are not seen in the corresponding wild type under the same conditions (Supplementary Fig. ). Across the range of metabolites measured, a core set of metabolites was seen to be significantly dependent on the level of thiamine supplementation (Fig. ). Notably, many of these metabolites were photosynthetic pigments. From the measurements of TDP levels in these seedlings, it appears that ca. 1 μmol g−1 FW (at the tissue level) is required for growth to approach wild-type levels (Fig. ), because stunting and chlorosis can still be observed with lower supplementation levels (Fig. ). These data therefore highlight the fact that TDP is needed in relatively small amounts but it is also evident that even a small deficit (low nM) severely reduces growth, likely by directly impacting photosynthesis, essentially halting development. The data thus demonstrate that TDP supply is essential for central metabolism and limitation of this coenzyme has major impacts on growth and development.

Abstract

Thiamin deficiency, or beriberi, is an increasingly re-recognized cause of morbidity and mortality in the developing world. Thiamin status has traditionally been measured through the erythrocyte activation assay (ETKA) or basal transketolase activity (ETK), which indirectly measure thiamin diphosphate (TDP). Thiamin diphosphate can also be measured directly by high-performance liquid chromatography (HPLC), which may allow a more precise estimation of thiamin status. We compared the direct measurement of TDP by HPLC with basal ETK activity and ETKA in 230 patients with Plasmodium falciparum malaria in rural southern Laos without overt clinical beriberi, as part of a trial of thiamin supplementation. Admission thiamin status measured by basal ETK activity and ETKA (α) were compared with thiamin status assessed by the measurement of TDP by HPLC. 55% of 230 included patients were male, and the median age was 10 (range 0.5–73) years. Using α ≥ 25% as the gold standard of thiamin deficiency, the sensitivity of TDP < 275 ng/gHb as a measure of thiamin deficiency was 68.5% (95% CI: 54.4–80.5%), with specificity of 60.8 (95% CI: 53.2–68.1%). There was a significant inverse correlation between the results of the two tests (Kendall’s tau = −0.212, P < 0.001). Basal ETK activity was also significantly positively correlated with TDP levels (Kendall’s tau = 0.576, P < 0.001). Thiamin diphosphate measurement may have a role in measuring thiamin levels in clinical settings. Further studies evaluating TDP concentration in erythrocytes with basal ETK activity and ETKA (α) in beriberi patients would help establish comparative values of these assays.

RESULTS

Using α ≥ 25% as the gold standard of biochemical thiamin deficiency, the sensitivity of erythrocyte TDP < 275 ng/gHb as a measure of thiamin deficiency was 68.5% (95% CI: 54.4–80.5%) with specificity of 60.8 (95% CI: 53.2–68.1%). There was a significant inverse correlation between the tests (Kendall’s tau = −0.212, P < 0.001). Inverse correlation was also observed between the two assays when adults () (78 patients, Kendall’s tau = −0.227, P = 0.003) and children () (152 patients, Kendall’s tau = −0.176, P = 0.001) were analyzed separately.

DISCUSSION

Limitations of this study include the fact that no patients in the cohort manifested clinical signs of thiamin deficiency, so it is not possible to correlate results with clinical thiamin deficiency. It is possible that some patients had subclinical deficiency. Determining a clinical endpoint for thiamin deficiency is challenging, as beriberi seems to occur not only when thiamin deficiency exists but also when additional exacerbating factors such as infections are present. The distinction between those who do and do not show clinical symptoms of beriberi at different biochemical assay levels may also be confounded by other factors. Importantly, these data cannot be used to inform biochemical diagnosis of infantile beriberi as children aged < 6 months were not included.

Abstract

The synthetic properties of the Thiamine diphosphate (ThDP)-dependent pyruvate dehydrogenase E1 subunit from Escherichia coli (EcPDH E1) was assessed for carboligation reactions with aliphatic ketoacids. Due to its role in metabolism, EcPDH E1 was previously characterised with respect to its biochemical properties, but it was never applied for synthetic purposes. Here, we show that EcPDH E1 is a promising biocatalyst for the production of chiral α-hydroxyketones. WT EcPDH E1 shows a 180–250-fold higher catalytic efficiency towards 2-oxobutyrate or pyruvate, respectively, in comparison to engineered transketolase variants from Geobacillus stearothermophilus (TKGST). Its broad active site cleft allows for the efficient conversion of both (R)- and (S)-configured α-hydroxyaldehydes, next to linear and branched aliphatic aldehydes as acceptor substrates under kinetically controlled conditions. The alternate, thermodynamically controlled self-reaction of aliphatic aldehydes was shown to be limited to low levels of conversion, which we propose to be due to their large hydration constants. Additionally, the thermodynamically controlled approach was demonstrated to suffer from a loss of stereoselectivity, which makes it unfeasible for aliphatic substrates.

2.1.2. Optimisation of Reaction Conditions

A spectrophotometric assay utilising 2,6-dichloroindophenol (DCPIP) and pyruvate as substrates () was initially used to identify optimal conditions for EcPDH E1 catalysed reactions []. The reduction in DCPIP was followed at its isosbestic point (517 nm, ) to allow for the determination of the optimal pH. E1 retained its activity over a broad pH range from pH 5.5 to 9.5, with its highest activity at pH 7.5 (a). The enzyme showed good thermal stability with no loss of activity after two hours of incubation at 40 °C. However, higher temperatures swiftly lead to its complete inactivation (b). Notably, EcPDH E1 can be stored with no loss of activity for at least six months at −20 °C (5 mg/mL EcPDH E1 in 20 mM potassium phosphate buffer, pH 7.0).