Novel regulatory mechanism of serine biosynthesis associated with 3-phosphoglycerate dehydrogenase in Arabidopsis thaliana

Okamura, Eiji; Hirai, Masami Yokota

doi:10.1038/s41598-017-03807-5

Novel regulatory mechanism of serine biosynthesis associated with 3-phosphoglycerate dehydrogenase in Arabidopsis thaliana

Article
Open access
Published: 14 June 2017

Volume 7, article number 3533, (2017)
Cite this article

Download PDF

You have full access to this open access article

Scientific Reports

Novel regulatory mechanism of serine biosynthesis associated with 3-phosphoglycerate dehydrogenase in Arabidopsis thaliana

Download PDF

Eiji Okamura¹ &
Masami Yokota Hirai¹

6433 Accesses
34 Citations
6 Altmetric
1 Mention
Explore all metrics

Abstract

The proteinogenic amino acid l-serine is a precursor for various essential biomolecules in all organisms. 3-Phosphoglycerate dehydrogenase (PGDH) is the first committed enzyme of the phosphorylated pathway of l-serine biosynthesis, and is regulated by negative feedback from l-serine in bacteria and plants. In the present study, two Arabidopsis PGDH isoforms were inhibited by l-serine but were activated by l-amino acids such as l-homocysteine in vitro. Activation and inhibition by these amino acids was cooperative, suggesting an allosteric mechanism. Moreover, the half maximal effective concentration of l-homocysteine was 2 orders of magnitude lower than that of l-serine, suggesting greater regulatory potency. These are the first data to show that PGDH is activated by various biomolecules and indicate that serine biosynthesis is regulated by multiple pathways.

Arabidopsis aldehyde dehydrogenase 10 family members confer salt tolerance through putrescine-derived 4-aminobutyrate (GABA) production

Article Open access 11 October 2016

Identification of Major Enzymes Involved in the Synthesis of Diadenosine Tetraphosphate and/or Adenosine Tetraphosphate in Myxococcus xanthus

Article 21 February 2018

Isolation and functional characterization of 3-phosphoglycerate dehydrogenase involved in salt responses in sugar beet

Article 26 May 2017

Introduction

In plants, l-serine is predominantly synthesized by glycolate and phosphorylated pathways. The glycolate pathway occurs in the mitochondria and is part of the photorespiratory pathway. This route is considered the main biosynthetic pathway of l-serine, at least in photosynthetic cells^{1, 2}. The phosphorylated pathway (Fig. 1a), which is found in mammals, bacteria, and plants, comprises reactions of 3-phosphoglycerate dehydrogenase (PGDH), phosphoserine aminotransferase, and phosphoserine phosphatase², and occurs in plastids³. The first committed enzyme PGDH oxidizes 3-phosphoglycerate (3-PGA), which originates from glycolysis and the Calvin cycle in heterotrophic and photosynthetic autotroph cells^{2, 4, 5}.

PGDH isoforms are classified according to protein domain structures⁶, and whereas PGDHs from organisms such as Sulfolobus tokodaii comprise only the catalytic domain⁷, other PGDHs possess an aspartate kinase–chorismate mutase–TyrA (ACT) domain and, in some cases, an allosteric substrate binding (ASB) domain (Fig. 1b)⁶. PGDH from Escherichia coli (EcPGDH) possesses only an additional ACT domain, and is allosterically and cooperatively inhibited by l-serine to achieve negative feedback regulation of the phosphorylated pathway⁸. Binding of l-serine to the ACT domain changes the conformation of EcPGDH and lowers its catalytic rate^{9, 10}. However, PGDH from Rattus norvegicus (RnPGDH) is considered to possess both ACT and ASB domains in addition to the catalytic domain, but is not inhibited by l-serine^{6, 11}, likely due to the reported absence of critical amino acid residues in the ACT domain⁶. PGDH from Mycobacterium tuberculosis (MtPGDH) comprises ACT, ASB, and catalytic domains and is cooperatively inhibited by l-serine in the presence of anions such as phosphate and chloride, and is non-cooperatively inhibited in the absence of these anions^{12, 13}. Depending on phosphate concentrations, MtPGDH forms homodimers, homotetramers, and homooctamers that vary in catalytic activity and sensitivity to inhibition by l-serine¹³. In active and l-serine-sensitive homotetramers and homooctamers, phosphate binds to the pocket formed by ASB and ACT domains of two PGDH molecules, and l-serine binds the ACT domain interface as previously reported^{6, 13, 14}. In contrast, PGDH from Aphanothece halophytica (AhPGDH) is not inhibited by l-serine, despite the presence of the putative ACT domain¹⁵. This indicates that the mechanism of allosteric inhibition by l-serine is not solely attributed to the presence of the ACT domain.

Arabidopsis thaliana expresses three PGDH isoforms and these are encoded by PGDH1 (At4g34200), PGDH2 (At1g17745), and PGDH3 (At3g19480)¹ (henceforth designated AtPGDH1, AtPGDH2, and AtPGDH3, respectively). Although AtPGDH1 and AtPGDH3, but not AtPGDH2, are inhibited by l-serine¹, the related modes of inhibition remain unknown. In plants, l-serine provides the carbon skeleton for the synthesis of l-cysteine and l-tryptophan, which are further converted into important biomolecules such as S-adenosyl-l-methionine (AdoMet) and the phytohormone indole acetic acid, respectively^{16, 17}. Therefore, we hypothesized that PGDH is regulated by multiple amino acids, as shown for aspartate kinase in the aspartate-family amino acid biosynthesis¹⁸, and determined the effects of various amino acids on AtPGDH activity in the presence and absence of l-serine.

In the present study, AtPGDH1 and AtPGDH3 were cooperatively activated by l-aspartate-derived l-homocysteine, l-methionine, and l-homoserine, by pyruvate-derived l-alanine and l-valine, and by l-serine-derived l-cysteine. Among these activators of AtPGDH, l-homocysteine had the lowest half maximal effective concentration (EC₅₀). The results suggested that the supply of l-serine is more refined than our conventional understanding in balancing the whole metabolic network.

Domain structures and biochemical properties of AtPGDHs

Multiple sequence alignments of AtPGDH1, AtPGDH2, and AtPGDH3 were performed with PGDHs from bacteria (EcPGDH, MtPGDH, and AhPGDH) and mammals (HsPGDH from Homo sapiens and RnPGDH from Rattus norvegicus; Fig. 2). All three Arabidopsis isoforms were previously shown to carry putative transit peptides for chloroplast localization^{1, 19, 20}, and the catalytic domain for oxidation of 3-PGA and concomitant reduction of NAD⁺ is located in N-terminal regions of all enzymes examined. Amino acid sequences of the catalytic domains of AtPGDHs have 42.2–44.8% identity with those of MtPGDH, whereas the C-terminal regions of PGDHs have low homology. However, all PGDHs except EcPGDH were predicted by InterPro²¹ to possess the ASB domain, and AtPGDHs were predicted to have ACT domains that are similar to AhPGDH, MtPGDH, and EcPGDH. The amino acid sequences of ASB and ACT domains of AtPGDHs had 15.6–17.9% and 19.1–21.9% identity to those of MtPGDH, respectively.

In this study, we expressed AtPGDH1, AtPGDH2, and AtPGDH3 genes heterologously in E. coli and analyzed the ensuing recombinant proteins using sodium dodecyl sulfate-polyacryamide gel electrophoresis (SDS-PAGE). These analyses indicated that all recombinant proteins were approximately 60 kDa, which was consistent with the theoretical molecular weights of AtPGDH1 (57.5 kDa), AtPGDH2 (57.6 kDa), and AtPGDH3 (57.9 kDa; Fig. 3a). Subsequent size exclusion chromatography analyses indicated that AtPGDH1 and AtPGDH3 predominantly form homotetramers, whereas AtPGDH2 formed an equilibrium of homooctamers and homotetramers under the present experimental conditions (Fig. 3b).

In the presence of 3-PGA as a substrate for l-serine synthesis, optimal pH values for the forward reaction were 9.0 (AtPGDH1), 10.0 (AtPGDH2), and 9.5 (AtPGDH3), respectively (Fig. 3c). This result is corroborated by the previous report stating that the optimal pH of PGDH purified from soybean root nodules was approximately 9.5²². Although AtPGDHs are localized to plastids¹, in which pH was shown to be 7.2²³, we conducted further biochemical experiments at pH 9.0. We determined apparent Michaelis constants (K _m ^app) and apparent maximum velocities (V _max ^app) of AtPGDH isoforms for 3-PGA and NAD⁺ (Fig. 3d). These values were similar among AtPGDH isoforms, indicating similar catalytic efficiencies of these isoforms at pH 9.0.

Some PGDHs, including MtPGDH, AhPGDH, and those from soybean root nodules, were activated by anions such as chloride in multiple previous studies^{12, 15, 22}, and all of the present AtPGDH isoforms were activated by chloride ions (Fig. 3e). To determine whether activation by chloride ions is cooperative, we plotted relative activities at various concentrations of chloride ions and fitted a sigmoidal E_max model with baseline (Hill equation)^{24, 25} to calculate Hill coefficients (n_H). In these computations, n_H = 1 indicates no cooperativity, and as summarized in Fig. 3e, Hill coefficients for activation were relatively small except for that for activation of AtPGDH2 by sodium chloride (2.3 ± 0.5), suggesting cooperativity.

2-Oxoglutarate is not a good substrate of AtPGDHs

Because PGDHs from E. coli and human reduce 2-oxoglutarate^{26, 27} and human PGDH also reduces oxaloacetate²⁷, we determined the reducing activities of the three AtPGDH isoforms with the substrates 2-oxoglutarate, oxaloacetate, and various carboxylic acids of the tricarboxylic acid (TCA) cycle (Fig. 4a). These analyses showed that only 2-oxoglutarate and oxaloacetate are substrates of AtPGDHs, and the optimal pH for reduction of 2-oxoglutarate and oxaloacetate was 6.0 (Fig. 4b). All AtPGDH isoforms showed Michaelis–Menten type curve at pH 6.0, with K _m ^app values that were 3–22 times higher than for 3-PGA (Fig. 4c). However, specificity constants k _cat/K _m of AtPGDH isoforms for 2-oxoglutarate and oxaloacetate were 6 orders of magnitude lower than those for 3-PGA, indicating that the three AtPGDH isoforms specifically oxidize 3-PGA for serine biosynthesis.

Novel cooperative activators of AtPGDHs

In previous studies, AtPGDH1 and AtPGDH3 activities were inhibited by serine¹, whereas AtPGDH2 activity was not affected by serine, threonine, valine, glycine, tryptophan, O-acetyl-l-serine, or cysteine^{1, 19}. In this study, we determined the effects of a total of 43 amino acids on AtPGDH activity at 10 mM (Fig. 5, Supplementary Fig. 1). Consistent with previous reports, l-serine inhibited AtPGDH1 and AtPGDH3 by 32% and 55%, respectively, but did not inhibit AtPGDH2. Moreover, l-alanine and l-valine, which are derived from pyruvate, significantly activated AtPGDH1 and AtPGDH3 (p < 0.001) by 140–170%. l-homoserine, l-homocysteine and l-methionine, which are derived from l-aspartate, also significantly activated these isoforms by 130–200%. However, d-form enantiomers of these amino acids did not activate AtPGDHs (Supplementary Fig. 1), indicating that these observations have physiological relevance. In contrast with AtPGDH1 and AtPGDH3, none of the present amino acids affected AtPGDH2 activity by more than 25% (p < 0.001). The sulfur-containing amino acids l-homocysteine and l-methionine activated AtPGDH1 and AtPGDH3. Because the sulfur atoms of these amino acids are derived from l-cysteine and the structures of l-serine and l-homocysteine are similar to that of l-cysteine, we performed further analyses to determine whether l-cysteine affects AtPGDH activity at higher concentrations.

To investigate modes of inhibition and activation by effector amino acids, relative activities of AtPGDHs were measured at various concentrations of effector amino acids and l-cysteine. Non-linear regression analyses of sigmoidal response curves of AtPGDH1 and AtPGDH3 revealed that absolute Hill coefficient values for l-serine, l-homocysteine, l-methionine, l-homoserine, l-alanine, and l-valine were greater than 2.0, indicating that these amino acids regulate AtPGDH activity in a cooperative manner (Fig. 6). Accordingly, whereas l- and d-cysteine were inhibitory, l-cysteine activated AtPGDH1 and AtPGDH3 at higher concentrations (Supplementary Fig. 2). Moreover, EC₅₀ values of the inhibitor l-serine for AtPGDH1 and AtPGDH3 were 6.6 and 1.3 mM, respectively, as shown previously¹. These values were much higher than those of l-serine for MtPGDH (approximately 30 µM in the presence of 150 mM NaCl)¹² and EcPGDH (approximately 8 µM)¹⁰, but were within the biological range of l-serine concentrations in chloroplasts (1 to >10 mM)^{28, 29}. Among activators of AtPGDH1 and AtPGDH3, l-homocysteine had the lowest EC₅₀ values, at 23 µM for AtPGDH1 and 69 µM for AtPGDH3. In contrast, EC₅₀ values of l-homoserine, l-alanine, and l-valine for AtPGDH1 and AtPGDH3 were in the mM order, and l-methionine affected AtPGDH1 activity at 0.67 mM. These data indicate that among effector amino acids, l-homocysteine is the most potent. Previously, l-serine, l-homoserine, l-alanine, l-valine, and l-methionine were present at 1.32, 1.52, 9.08, 1.36, and 0.12 mM, respectively, in chloroplasts from pea leaves²⁹. In addition, ratios of pool sizes of methionine and homocysteine in Chlorella sorokiniana indicated that homocysteine was present at 0.47 mM³⁰. Accordingly, the present EC₅₀ values for AtPGDH1 and AtPGDH3 are within the biological ranges of these amino acid concentrations.

Domains required for regulation by effector amino acids

The ACT domain is considered as the amino acid-binding site in enzymes that are regulated by amino acids³¹. Thus, to confirm the site of amino acid binding, we swapped ACT domain of amino acid-sensitive AtPGDH1 with that of the insensitive AtPGDH2. In these experiments, the N-terminal half (containing the catalytic domain) of AtPGDH2 (designated as N2) was fused to the C-terminal half (containing ASB and ACT domains) of AtPGDH1 (designated as C1), and vice versa to construct AtPGDH-N2/C1 and AtPGDH-N1/C2 (Fig. 7a). Subsequent size exclusion chromatography analyses suggested that AtPGDH-N2/C1 forms homotetramers (Fig. 7b), whereas AtPGDH-N1/C2 was mainly found as a homotetramer, and as multimers of to up to 20 subunits. The analyses of wild-type and chimeric enzymes indicate that the C-terminal half of AtPGDH2 is involved in multimerization beyond tetramers. Kinetic parameters of these chimeric enzymes were comparable with those of wild-type enzymes, with k _cat/K _m (M⁻¹s⁻¹) values of 0.9 × 10⁴ (3-PGA) and 0.73 × 10⁵ (NAD⁺), and 1.3 × 10⁴ (3-PGA) and 0.46 × 10⁵ (NAD⁺) for AtPGDH-N2/C1 and AtPGDH-N1/C2, respectively (Fig. 7c), and contrasting values of 1.6 × 10⁴ (3-PGA) and 1.3 × 10⁵ (NAD⁺) and 3.0 × 10⁴ (3-PGA) and 3.0 × 10⁵ (NAD⁺) for AtPGDH2 and AtPGDH1, respectively (Fig. 3). These results indicate that specific activities of these catalytic domains are not remarkably affected in these constructs.

We determined changes in specific activities of the chimeric enzymes following treatments with effector amino acids (Fig. 8). In these experiments, AtPGDH-N2/C1, which carries ASB and ACT domains from the amino acid-sensitive AtPGDH1, was cooperatively inhibited by l-serine, with a similar EC₅₀ value to that of AtPGDH1. However, AtPGDH-N1/C2, which carries ASB and ACT domains from the amino acid-insensitive AtPGDH2, was not inhibited by l-serine. Considering the likely function of the ACT domain as an amino acid-binding site, these results suggest that l-serine binds the ACT domain of AtPGDH1, and that the C-terminal region is involved in cooperativity.

Other effector amino acids significantly activated AtPGDH-N2/C1, but with lower potency than with AtPGDH1 (Figs 7d and 8), suggesting that l-homocysteine, l-methionine, l-homoserine, and l-valine bind the C1 region. However, some features of the N1 region were required for full activation by these amino acids. l-alanine cooperatively activated both AtPGDH-N2/C1 and AtPGDH-N1/C2, with EC₅₀ values that were similar to those of AtPGDH1. The data suggests that either N1 or C1 regions are sufficient for cooperative activation of AtPGDH1 by l-alanine. Moreover, consistent with the other activators, both N1 and C1 regions were necessary for full activation. In summary, these results suggest that cooperative activation mechanisms by effector amino acids require the formation of higher order structures of C- and N-terminal half regions via intra- and/or inter-molecular interactions. Moreover, this mechanism may reflect the formation of tetramers from enzymes with the C1 region, compared with the formation of multimers of proteins with the C2 region (Figs 3 and 7).

Combined effects of l-serine and amino acid activators

Because l-serine and the present activator amino acids coexist in vivo, we compared dose responses to l-serine in the presence of various activators in vitro. The EC₅₀ value of l-serine for AtPGDH1 was shifted to higher concentrations in the presence of activators, although maximum inhibitory effects of l-serine were relatively unchanged (Fig. 9a). In contrast, the effect of 20 mM l-serine was decreased with increasing activator concentrations (Fig. 9b), and l-homocysteine almost completely abolished inhibition of AtPGDH1 by l-serine at concentrations of greater than 0.1 mM. Moreover, the other activators prevented inhibition of AtPGDH1 by l-serine to varying degrees, and the effects of l-homocysteine were greatest, followed by those of l-methionine. As shown for AtPGDH3, l-methionine, l-valine, l-alanine, and l-homoserine did not fully abolish inhibition by l-serine at the present activator concentrations.

Discussion

The present chimeric enzyme experiments suggest that l-serine binds the ACT domain of AtPGDH1, and that the C-terminal region mediates cooperativity of inhibition by l-serine. MtPGDH possesses the same domain architecture as AtPGDHs and is sensitive to inhibition by l-serine, and Y461, D463, and N481 residues of the ACT domain were shown to be central to l-serine binding¹⁴. In AtPGDHs, the residues that correspond to D463 and N481 are conserved regardless of sensitivity to l-serine. These residues are D538 and N556 in AtPGDH1, D559 and N577 in AtPGDH2, and D523 and N541 in AtPGDH3 (Fig. 2). Although Y461 is not conserved in AtPGDHs, the corresponding residue is Q in all three isoforms. Taken together, these observations suggest that other factors play roles in differences in l-serine sensitivity between AtPGDH2 and the other two isoforms. Crystal structure analyses³² show that homotetramers of MtPGDH are asymmetric, reflecting the presence of the intervening domain that corresponds to the ASB domain. Moreover, the formation of one of two conformations of the regulatory domain (corresponding to the ACT domain) on each subunit likely leads to differing accessibility of l-serine. In agreement, whereas AtPGDH2 was present in an equilibrium of homotetramers and homooctamers, AtPGDH1 and AtPGDH3 only formed homotetramers (Fig. 3). Recently, a novel allosteric binding Site I was identified in EcPGDH³³, and an ASB-homologous domain in serine dehydratase was suggested to bind the substrate serine³⁴. Thus, further structural analysis of AtPGDHs may lead to the discovery of higher structures that are responsible for allosteric regulation of PGDHs.

The AtPGDH1 gene was previously identified as the embryo sac development arrest 9 gene, and its mutation was embryonically lethal³⁵. This gene also plays an important role in pollen development²⁰. Other studies showed that the AtPGDH1 gene is coexpressed with genes of tryptophan biosynthesis¹, and that the activators of tryptophan biosynthesis MYB51 and MYB34 are involved in the expression of AtPGDH1 ¹. Thus, we think that AtPGDH1 is responsible for the supply of serine as a substrate for tryptophan synthase (Fig. 10). In fact, tryptophan-derived metabolites such as indole acetic acid and indole glucosinolates were present at lower concentrations in AtPGDH1-silenced plants¹, demonstrating that serine used as a precursor for tryptophan biosynthesis is synthesized in the phosphorylated pathway, and that the involvement of AtPGDH1 is greatest among the three isoforms. On the other hand, l-serine is also a precursor for sulfur-containing amino acids, and is converted into O-acetyl-l-serine and then reacts with sulfide to form l-cysteine (Fig. 10). These two reactions are catalyzed by serine acetyltransferase and O-acetylserine (thiol) lyase, and are finely regulated at the enzyme activity level depending on the availability of sulfur¹⁶. Hence, the positive and sensitive regulation of AtPGDH1 and AtPGDH3 by l-homocysteine may contribute to the balance between sulphur assimilation and tryptophan biosynthesis. l-homocysteine is converted into AdoMet via l-methionine in the S-adenosyl-l-methionine cycle^{36, 37}. The conversion of l-homocysteine to l-methionine is associated with folate-mediated one-carbon metabolism, in which single carbon units originate from l-serine and other molecules³⁸. AdoMet is converted into S-adenosyl-l-homocysteine and then into l-homocysteine³⁶. Hence, l-homocysteine may be a signaling molecule that enhances AdoMet production by activating l-serine biosynthesis.

During photosynthesis, ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a key enzyme for carbon fixation via the Calvin cycle and forms 3-PGA by reacting with carbon dioxide (CO₂; Fig. 10). Photorespiration is initiated by binding of oxygen to Rubisco as a substrate instead of CO₂, resulting in glycolate formation. Accordingly, under high CO₂ conditions where photorespiration is repressed, CO₂ fixation is enhanced. In agreement, valine, alanine, leucine, and histidine accumulations were enhanced in wild-type A. thaliana in the presence of high CO₂ concentrations¹. Under these CO₂ conditions, serine biosynthesis via the glycolate pathway is repressed, likely increasing the importance of the phosphorylated pathway. In accordance, rosette leaf development was completely repressed by high CO₂ conditions in AtPGDH1-silenced A. thaliana and AtPGDH1 expression was induced¹, suggesting that AtPGDH1 is essential for plant adaptation to high CO₂, and to embryogenesis and pollen development. In the present experiments, AtPGDH1 was activated by valine and alanine, which are present at increased concentrations under conditions of high CO₂. Thus, serine biosynthesis via AtPGDH1 seems to be enhanced under high CO₂ conditions, reflecting both transcriptional and enzymatic activation.

The mitochondrial glycolate pathway is generally considered the major source of serine in photosynthetic organs, whereas the plastid phosphorylated pathway is predominant in non-photosynthetic organs². However, the relative contributions of these pathways to serine biosynthesis in various organs and at various developmental stages remain unclear. In addition, the interplay between these pathways has not been investigated. In a recent study, exogenously supplied serine induced the expression of AtPGDH2 and AtPGDH1 in aerial parts and roots and in aerial parts only, respectively³⁹. These observations are in apparent contradiction with regulatory enzymatic feedback inhibition by serine. Moreover, the AtPGDH activating mechanisms shown herein further indicate greater regulatory intricacies of metabolic networks than those based purely on the regulation of the phosphorylated pathway by serine.

Methods

Multiple alignments of amino acid sequences

Amino acid sequences of PGDHs from A. thaliana (AtPGDHs), A. halophytica (AhPGDH), M. tuberculosis (MtPGDH), R. norvegicus (RnPGDH), H. sapiens (HsPGDH), and E. coli (EcPGDH) were aligned using CLUSTAL W with MEGA6 software^{40, 41}. Alignments were rendered using ESPript (http://espript.ibcp.fr)^{42, 43}.

Accession numbers for each amino acid sequence are as follows: AtPGDH1, NP_195146 (EMBL/GenBank/DDBJ); AtPGDH2, NP_564034 (EMBL/GenBank/DDBJ); AtPGDH3, NP_566637 (EMBL/GenBank/DDBJ); AhPGDH, BAF91727.1 (EMBL/GenBank/DDBJ); MtPGDH, 1YGY chain A (PDB); RnPGDH, CAA66374.1 (EMBL/GenBank/DDBJ); HsPGDH, NP_006614.2 (EMBL/GenBank/DDBJ) and EcPGDH 1YBA chain A (PDB).

Reagents

Nicotine amide adenine dinucleotide (NAD) disodium salt, 3-phosphoglycerate [D-(–)-3-phosphoglyceric acid disodium salt], l-homoserine, d-homoserine, l-homocysteine, 2-oxoglutarate (α-ketoglutaric acid sodium salt), oxaloacetate, cis-aconitate, molecular weight standards (Gel Filtration Markers Kit for Protein Molecular Weights) for size exclusion chromatography analyses, and Murashige–Skoog (MS) vitamin solution were purchased from Sigma–Aldrich Co., Ltd. (St. Louis, MO, USA). The other amino acids, the salt mixture of MS medium, Good’s buffers, NADH (β-diphosphopyridine nucleotide disodium salt, reduced form), fumarate, malate (l-malate), citrate (citric acid monohydrate), and isocitrate [trisodium ( ± )-isocitrate n-hydrate] were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

Plant materials

To clone AtPGDH3 (At3g19480) cDNA, A. thaliana accession Columbia-0 (Col-0) were grown on agar-solidified half-strength MS medium containing 0.5% sucrose, 1 mM MES, and 0.1% MS vitamin solution (pH 5.8) for 3 weeks under fluorescent light/dark conditions (16-h light/8-h dark) at 22 °C.

Preparation of recombinant enzymes

Full-length cDNA clones of AtPGDH1 (At4g34200) and AtPGDH2 (At1g17745) were obtained from RIKEN Bio Resource Center (accession code pda02295 and pda04481, respectively)^{44, 45}. Full-length cDNA of AtPGDH3 (At3g19480) was synthesized using a PrimeScript II High Fidelity One Step RT-PCR Kit (Takara Bio Inc., Kyoto, Japan) with total RNA from 3-week-old A. thaliana rosette leaves using the RNeasy Plant mini Kit (Qiagen) as a template. Subsequently, cDNA was amplified using the primers shown in Supplementary Table 1 and were directly inserted into pENTR-D-TOPO vectors (Life Technologies) and then sequenced.

Transit peptide sequences for chloroplast localization of AtPGDH1, AtPGDH2, and AtPGDH3 were predicted by comparing their amino acid sequences with that of the MtPGDH gene. Regions that correspond with mature enzymes were amplified using the primers shown in Supplementary Table 1 and were then inserted into SpeI and NotI sites of the expression vector pPAL7 (Bio-RAD) using In-Fusion cloning with the In-Fusion HD cloning kit (Takara Bio Inc.)⁴⁶.

Recombinant enzymes were expressed in E. coli BL21 CodonPlus (DE3)-RIL (Agilent). Pre-cultivation was performed in Luria–Bertani (LB) liquid medium containing 100 µg/mL carbenicillin and 30 µg/mL chloramphenicol at 28 °C for 12 h. Subsequently, 2% cultures were inoculated into 150 mL of LB liquid medium containing 50 µg/mL carbenicillin and 30 µg/mL chloramphenicol and cells were grown at 20 °C until OD₆₀₀ reached 0.5. Subsequently, isopropyl β-d-thiogalactopyranoside was added (final concentration 0.5 mM) and cells were incubated for a further 12 h at 20 °C.

Tag-free recombinant proteins were prepared using a Profinity eXact Fusion-Tag System (Bio-RAD) and a Profinia Protein Purification instrument (Bio-RAD) equipped with Bio-Scale Mini Profinity eXact Cartridges (1 mL) for affinity purification. Bio-Scale Mini Bio-Gel P-6 Desalting Cartridges (10 mL) were used for desalting according to the manufacturer’s instructions. Briefly, cell pellets were obtained by centrifugation of E. coli cultures at 9,000 × g and were then suspended in 100 mM sodium phosphate buffer (pH 9.0) and sonicated on ice for 10 min. Crude extracts were then centrifuged for 10 min at 9,000 × g. Supernatants were then applied to a Profinia Protein Purification Instrument and on-column incubations were performed for 1 h at 20 °C to eliminate the affinity tag eXact Fusion-Tag from recombinant proteins. Eluted fractions were then immediately concentrated to 250 µL by ultrafiltration using an Amicon Ultra-4 Centrifugal filter Unit (MWCO 10,000; Merck-Millipore).

Domain swapping of AtPGDHs was performed as follows. Initially, regions of ASB and ACT domains from AtPGDHs were estimated using Uniport⁴⁷ (Fig. 2). N-terminal half regions containing catalytic domains and C-terminal half regions containing ASB and ACT domains were then amplified using specific primer sets (Supplementary Table 1). Amplified N- and C-terminal fragments were then inserted into SpeI and NotI sites of the expression vector pPAL7 with combinations of AtPGDH-N1 and AtPGDH-C2 or AtPGDH-N2 and AtPGDH-C1, and homologous recombination was performed using an In-Fusion HD cloning kit⁴⁶.

Estimation of molecular weights of recombinant AtPGDHs

Molecular weights of wild-type and chimeric AtPGDHs in native states were estimated in size exclusion chromatography analyses (flow rate 1 mL/min) in the presence of 100 mM phosphate using AKTA explorer 10 s (for AtPGDH1, AtPGDH2 and AtPGDH3) or AKTA Start (for AtPGDH-2N/1 C and AtPGDH-1N/2 C) instruments with HiLoad 26/600 Superdex 200 preparation grade columns (GE Healthcare). Molecular weights were calculated based on standard curves of thyroglobulin (669 kDa), apoferritin (443 kDa), β-amylase (200 kDa), alcohol dehydrogenase (150 kDa), albumin (66 kDa), and carbonic anhydrase (29 kDa) in 100 mM sodium phosphate buffer (pH 9.0).

Spectrophotometric assays of recombinant AtPGDHs

3-PGA oxidation activity was assayed in 600 µL reaction mixtures containing 0.1 M Good’s buffer (described below), 1 mM dithiothreitol, 10 mM 3-PGA, 1 mM NAD⁺, 0.1 M NaCl, and 5.0 µg of recombinant enzyme. After preincubation of reaction mixtures without 3-PGA at 25 °C for 10 min, reactions were initiated by adding 3-PGA^{12, 48}. To determine substrate specificities of reduction activity, 10 mM carboxylic acid solutions (citrate, cis-aconitate, isocitrate, oxaloacetate, succinate, fumarate, malate, or 2-oxoglutarate) were incubated in 600 µL reaction mixtures containing 0.1 M TAPS (pH 9.0), 1 mM dithiothreitol, 0.1 mM NADH, 0.1 M NaCl, and 10 µg of recombinant enzyme. Subsequently, 3-PGA oxidation activities were determined according to increases in absorbance of NADH (340 nm), which is generated from NAD⁺ with the oxidation of 3-PGA. Conversely, carboxylic acid reduction was determined by monitoring decreases in absorbance at 340 nm using a UV-2700 spectrophotometer (Shimadzu, Kyoto, Japan). Reaction mixtures without substrates were used as negative controls, and changes in absorbance at 340 nm were monitored under the same conditions. Experiments were performed 2 times. In some cases, experimental conditions differed slightly and single representative results are presented.

Optimal pH values for 3-PGA oxidation activity were determined in the presence of 10 mM 3-PGA and 1 mM NAD⁺ at pH intervals of 0.5 between 6.0 and 11.0. Good’s buffers were used as follows: MES-NaOH for pH 6.0 and 6.5; HEPES-NaOH for pH 7.0, 7.5, and 8.0; TAPS-NaOH for pH 8.5 and 9.0; CHES-NaOH for pH 9.5 and 10.0, and CAPS-NaOH for pH 10.5 and 11.0. Data were collected from three technical replicates. The optimal pH for reduction of 2-oxoglutarate and oxaloacetate was determined using 10 mM 2-oxoglutarate or oxaloacetate and 0.1 mM NADH at pH intervals of 0.5 between 6.0 and 9.0.

To determine kinetic parameters for 3-PGA oxidation activity at pH 9.0, 3-PGA was added at 0.1, 0.2, 0.5, 0.7, 1, 2, 5, 7, and 10 mM with a fixed NAD⁺ concentration of 1 mM. In separate experiments, NAD⁺ was applied at 0.01, 0.02, 0.05, 0.07, 0.1, 0.2, 0.5, 0.7, and 1.0 mM with a fixed 3-PGA concentration of 10 mM. Initial velocities were determined from slopes of plots of NADH formation versus incubation time. To determine kinetic parameters for the reduction of 2-oxoglutarate and oxaloacetate at pH 6.0, 2-oxoglutarate and oxaloacetate were added at 1, 2, 5, 7, 10, and 20 mM with a fixed NADH concentration of 0.1 mM. Initial velocities were determined from slopes of plots of decreases in NADH versus incubation time. The molar extinction coefficient (ε) of NADH at 340 nm was 6.2 × 10³. Kinetic parameters [apparent Michaelis constants (K _m ^app) and apparent maximum velocities (V _max ^app)] were calculated by fitting specific activities (v) to Michaelis-Menten equations under various concentrations of substrate (S)⁴⁹ as follows:

$$v={V}_{max}{\rm{S}}/({K}_{{\rm{m}}}+{\rm{S}})$$

where V _max and K _m represent the maximal velocities and the Michaelis constants, respectively. Data fitting was performed using the Enzyme kinetics module in SigmaPlot (Systat Software, San Jose, CA).

To determine whether chloride ions activate 3-PGA oxidation activity, specific activities were determined at 10 mM 3-PGA and 1 mM NAD⁺ in the presence of 0, 10, 20, 50, 70, 100, 200, and 500 mM NaCl or KCl. Data were collected from three technical replicates.

Sensitivity to various amino acids was tested using the aforementioned method with slight modifications. Initially, the effects of amino acids were determined at 10 mM by calculating specific activities of the enzyme. Subsequently, specific activities were determined at 10 mM 3-PGA and 1 mM NAD⁺ in the presence of various concentrations of amino acids as follows: for AtPGDH1, AtPGDH2, and AtPGDH3, l-homoserine, l-alanine, l-valine, l-cysteine, and d-cysteine were added at 0, 0.1, 0.2, 0.5, 0.7, 1, 2, 5, 7, 10, 20, and 50 mM; for AtPGDH1, l-methionine was added at 0, 0.01, 0.02, 0.05, 0.07, 0.1, 0.2, 0.5, 0.7, 1, 2, 5, 7, 10, 20, and 50 mM; for AtPGDH2, l-methionine was added at 0, 0.1, 0.2, 0.5, 0.7, 1, 2, 5, 7, 10, 20, and 50 mM; for AtPGDH3, l-methionine was added at 0, 0.1, 0.2, 0.5, 0.7, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 7, 10, 20, and 50 mM; for AtPGDH1 and AtPGDH3, l-serine was added at 0, 0.1, 0.2, 0.5, 0.7, 1, 2, 5, 7, 10, 20, and 50 mM; for AtPGDH2, l-serine was added at 0, 0.1, 0.2, 0.5, 0.7, 1, 2, 5, 7, and 10 mM; for AtPGDH1, l-homocysteine was added at 0, 0.001, 0.002, 0.005, 0.007, 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.07, 0.1, 0.2, 0.5, 0.7, and 1 mM; for AtPGDH2, l-homocysteine was added at 0, 0.001, 0.002, 0.005, 0.007, 0.01, 0.02, 0.05, 0.07, 0.1, 0.2, 0.5, 0.7, and 1 mM; for AtPGDH3 l-homocysteine was added at 0, 0.001, 0.002, 0.005, 0.007, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.5, 0.7, and 1 mM. To determine the effects of amino acids on AtPGDH-N2/C1 and AtPGDH-N1/C2 activities, ranges of amino acid concentrations were set as follows: l-serine, l-valine, l-homoserine, and l-methionine, 0, 0.1, 0.2, 0.5, 0.7, 1, 2, 5, 7, 10, 20, and 50 mM; l-homocysteine, 0, 0.001, 0.002, 0.005, 0.007, 0.01, 0.02, 0.05, 0.07, 0.1, 0.2, 0.5, 0.7, and 1 mM; l-alanine, 0, 0.1, 0.2, 0.5, 0.7, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 7, 10, 20, and 50 mM. To determine combined effects of activators in the presence of l-serine, ranges of amino acid concentrations were set as follows: l-methionine, l-valine, l-homoserine, and l-alanine, 0, 0.1, 0.2, 0.5, 0.7, 1, 2, 5, 7, 10, 20, and 50 mM; l-homocysteine, 0.01, 0.02, 0.05, 0.07, 0.1, 0.2, 0.5, 0.7, 1, 2, and 5 mM.

Non-linear regression with Hill equations

Cooperativities and half maximal effective concentrations (EC₅₀) of effectors were determined by fitting percentage relative activities (the specific activity at 0 mM was set at 100) of effectors (E) at various effector concentrations (C) to the sigmoidal E_max model with baseline response (the Hill equation)²⁴ as follows:

$${\rm{E}}={{\rm{E}}}_{0}+{{\rm{E}}}_{{\rm{\max }}}{{\rm{C}}}^{{{\rm{n}}}_{{\rm{H}}}}/({{{\rm{EC}}}_{50}}^{{{\rm{n}}}_{{\rm{H}}}}+{{\rm{C}}}^{{{\rm{n}}}_{{\rm{H}}}})$$

where E₀ represents the relative activity in the absence of effector (baseline), E_max represents the relative activity at the maximal effective concentration (inhibition or activation) of the effector, n_h is the Hill coefficient, and EC₅₀ is half maximal effective concentration of the effector at half E_max.

The parameters E₀, E_max, n_H, and EC₅₀ were estimated using three or six data sets for percentage relative activities (E) under various effector concentrations (C). The data were then fitted to the sigmoidal E_max model with baseline response using the nonlinear least squares method (Levenberg-Marquardt algorithm) without weight functions using SigmaPlot. In the tables, EC₅₀ and n_h values are shown as estimated values ± standard errors only when p values for n_h are smaller than 0.015.

References

Benstein, R. M. et al. Arabidopsis phosphoglycerate dehydrogenase1 of the phosphoserine pathway is essential for development and required for ammonium assimilation and tryptophan biosynthesis. Plant Cell. 25, 5011–5029 (2013).
Article CAS PubMed PubMed Central Google Scholar
Ros, R., Munoz-Bertomeu, J. & Krueger, S. Serine in plants: biosynthesis, metabolism, and functions. Trends Plant Sci. 19, 564–569 (2014).
Article CAS PubMed Google Scholar
Ho, C. L. & Saito, K. Molecular biology of the plastidic phosphorylated serine biosynthetic pathway in Arabidopsis thaliana. Amino Acids 20, 243–259 (2001).
Article CAS PubMed Google Scholar
Hausler, R. E., Ludewig, F. & Krueger, S. Amino acids–a life between metabolism and signaling. Plant Sci. 229, 225–237 (2014).
Article PubMed Google Scholar
Ros, R. et al. Serine biosynthesis by photorespiratory and non-photorespiratory pathways: an interesting interplay with unknown regulatory networks. Plant Biol (Stuttg) 15, 707–712 (2013).
Article CAS Google Scholar
Grant, G. A. Contrasting catalytic and allosteric mechanisms for phosphoglycerate dehydrogenases. Arch. Biochem. Biophys. 519, 175–185 (2012).
Article CAS PubMed Google Scholar
Shimizu, Y., Sakuraba, H., Doi, K. & Ohshima, T. Molecular and functional characterization of D-3-phosphoglycerate dehydrogenase in the serine biosynthetic pathway of the hyperthermophilic archaeon Sulfolobus tokodaii. Arch. Biochem. Biophys. 470, 120–128 (2008).
Article CAS PubMed Google Scholar
Thompson, J. R., Bell, J. K., Bratt, J., Grant, G. A. & Banaszak, L. J. Vmax regulation through domain and subunit changes. The active form of phosphoglycerate dehydrogenase. Biochemistry. 44, 5763–5773 (2005).
Article CAS PubMed Google Scholar
Chipman, D. M. & Shaanan, B. The ACT domain family. Curr. Opin. Struct. Biol. 11, 694–700 (2001).
Article CAS PubMed Google Scholar
Sugimoto, E. & Pizer, L. I. The mechanism of end product inhibition of serine biosynthesis. I. Purification and kinetics of phosphoglycerate dehydrogenase. J. Biol. Chem. 243, 2081–2089 (1968).
CAS PubMed Google Scholar
Achouri, Y., Rider, M. H., Schaftingen, E. V. & Robbi, M. Cloning, sequencing and expression of rat liver 3-phosphoglycerate dehydrogenase. Biochem. J. 323(Pt 2), 365–370 (1997).
Article CAS PubMed PubMed Central Google Scholar
Dey, S., Hu, Z., Xu, X. L., Sacchettini, J. C. & Grant, G. A. D-3-Phosphoglycerate dehydrogenase from Mycobacterium tuberculosis is a link between the Escherichia coli and mammalian enzymes. J. Biol. Chem. 280, 14884–14891 (2005).
Article CAS PubMed Google Scholar
Xu, X. L. & Grant, G. A. Regulation of Mycobacterium tuberculosis D-3-phosphoglycerate dehydrogenase by phosphate-modulated quaternary structure dynamics and a potential role for polyphosphate in enzyme regulation. Biochemistry. 53, 4239–4249 (2014).
Article CAS PubMed Google Scholar
Dey, S., Burton, R. L., Grant, G. A. & Sacchettini, J. C. Structural analysis of substrate and effector binding in Mycobacterium tuberculosis D-3-phosphoglycerate dehydrogenase. Biochemistry. 47, 8271–8282 (2008).
Article CAS PubMed PubMed Central Google Scholar
Waditee, R. et al. Metabolic engineering for betaine accumulation in microbes and plants. J. Biol. Chem. 282, 34185–34193 (2007).
Article CAS PubMed Google Scholar
Takahashi, H., Kopriva, S., Giordano, M., Saito, K. & Hell, R. Sulfur assimilation in photosynthetic organisms: molecular functions and regulations of transporters and assimilatory enzymes. Annu Rev Plant Biol. 62, 157–184 (2011).
Article CAS PubMed Google Scholar
Radwanski, E. R. & Last, R. L. Tryptophan biosynthesis and metabolism: biochemical and molecular genetics. Plant Cell. 7, 921–934 (1995).
Article CAS PubMed PubMed Central Google Scholar
Jander, G. & Joshi, V. Aspartate-derived amino acid biosynthesis in Arabidopsis thaliana. Arabidopsis Book 7, e0121, doi:10.1199/tab.0121 (2009).
Article PubMed PubMed Central Google Scholar
Ho, C. L., Noji, M., Saito, M. & Saito, K. Regulation of serine biosynthesis in Arabidopsis. Crucial role of plastidic 3-phosphoglycerate dehydrogenase in non-photosynthetic tissues. J. Biol. Chem. 274, 397–402 (1999).
Article CAS PubMed Google Scholar
Toujani, W. et al. Functional characterization of the plastidial 3-phosphoglycerate dehydrogenase family in Arabidopsis. Plant Physiol. 163, 1164–1178 (2013).
Article CAS PubMed PubMed Central Google Scholar
Mitchell, A. et al. The InterPro protein families database: the classification resource after 15 years. Nucleic Acids Res. 43, D213–D221 (2015).
Article PubMed Google Scholar
Boland, M. J. & Schubert, K. R. Phosphoglycerate dehydrogenase from soybean nodules: partial purification and some kinetic properties. Plant Physiol. 71, 658–661 (1983).
Article CAS PubMed PubMed Central Google Scholar
Shen, J. et al. Organelle pH in the Arabidopsis endomembrane system. Mol Plant 6, 1419–1437 (2013).
Article CAS PubMed Google Scholar
Goutelle, S. et al. The Hill equation: a review of its capabilities in pharmacological modelling. Fundam. Clin. Pharmacol. 22, 633–648 (2008).
Article CAS PubMed Google Scholar
Hill, A. V. The possible effects of the aggregation of the molecules of hæmoglobin on its dissociation curves. J Physiol. 40, iv–vii (1910).
Google Scholar
Zhao, G. & Winkler, M. E. A novel alpha-ketoglutarate reductase activity of the serA-encoded 3-phosphoglycerate dehydrogenase of Escherichia coli K-12 and its possible implications for human 2-hydroxyglutaric aciduria. J. Bacteriol. 178, 232–239 (1996).
Article CAS PubMed PubMed Central Google Scholar
Fan, J. et al. Human phosphoglycerate dehydrogenase produces the oncometabolite D-2-hydroxyglutarate. ACS Chem Biol. 10, 510–516 (2015).
Article CAS PubMed Google Scholar
Riens, B., Lohaus, G., Heineke, D. & Heldt, H. W. Amino Acid and sucrose content determined in the cytosolic, chloroplastic, and vacuolar compartments and in the phloem sap of spinach leaves. Plant Physiol. 97, 227–233 (1991).
Article CAS PubMed PubMed Central Google Scholar
Mills, W. R. & Joy, K. W. A rapid method for isolation of purified, physiologically active chloroplasts, used to study the intracellular distribution of amino acids in pea leaves. Planta. 148, 75–83 (1980).
Article CAS PubMed Google Scholar
Giovanelli, J., Mudd, S. H. & Datko, A. H. Homocysteine biosynthesis in green plants. Physiological importance of the transsulfuration pathway in Chlorella sorokiniana growing under steady state conditions with limiting sulfate. J. Biol. Chem. 253, 5665–5677 (1978).
CAS PubMed Google Scholar
Grant, G. A. The ACT domain: a small molecule binding domain and its role as a common regulatory element. J. Biol. Chem. 281, 33825–33829 (2006).
Article CAS PubMed Google Scholar
Dey, S., Grant, G. A. & Sacchettini, J. C. Crystal structure of Mycobacterium tuberculosis D-3-phosphoglycerate dehydrogenase: extreme asymmetry in a tetramer of identical subunits. J. Biol. Chem. 280, 14892–14899 (2005).
Article CAS PubMed Google Scholar
Wang, Q., Qi, Y., Yin, N. & Lai, L. Discovery of novel allosteric effectors based on the predicted allosteric sites for Escherichia coli D-3-phosphoglycerate dehydrogenase. PLoS One 9, e94829, doi:10.1371/journal.pone.0094829 (2014).
Article ADS PubMed PubMed Central Google Scholar
Grant, G. A. Kinetic evidence of a noncatalytic substrate binding site that regulates activity in Legionella pneumophila L-serine dehydratase. Biochemistry. 51, 6961–6967 (2012).
Article CAS PubMed Google Scholar
Pagnussat, G. C. et al. Genetic and molecular identification of genes required for female gametophyte development and function in Arabidopsis. Development. 132, 603–614 (2005).
Article CAS PubMed Google Scholar
Sauter, M., Moffatt, B., Saechao, M. C., Hell, R. & Wirtz, M. Methionine salvage and S-adenosylmethionine: essential links between sulfur, ethylene and polyamine biosynthesis. Biochem. J. 451, 145–154 (2013).
Article CAS PubMed Google Scholar
Ravanel, S. et al. Methionine metabolism in plants: chloroplasts are autonomous for de novo methionine synthesis and can import S-adenosylmethionine from the cytosol. J. Biol. Chem. 279, 22548–22557 (2004).
Article CAS PubMed Google Scholar
Hanson, A. D., Gage, D. A. & Shachar-Hill, Y. Plant one-carbon metabolism and its engineering. Trends Plant Sci. 5, 206–213 (2000).
Article CAS PubMed Google Scholar
Toujani, W. et al. Identification of the phosphoglycerate dehydrogenase isoform EDA9 as the essential gene for embryo and male gametophyte development in Arabidopsis. Plant Signal Behav. 8, e27207, doi:10.4161/psb.27207 (2013).
Article PubMed PubMed Central Google Scholar
Thompson, J. D., Higgins, D. G. & Gibson, T. J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680 (1994).
Article CAS PubMed PubMed Central Google Scholar
Tamura, K., Stecher, G., Peterson, D., Filipski, A. & Kumar, S. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30, 2725–2729 (2013).
Article CAS PubMed PubMed Central Google Scholar
Gouet, P., Courcelle, E., Stuart, D. I. & Metoz, F. ESPript: analysis of multiple sequence alignments in PostScript. Bioinformatics. 15, 305–308 (1999).
Article CAS PubMed Google Scholar
Robert, X. & Gouet, P. Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res. 42, W320–W324 (2014).
Article CAS PubMed PubMed Central Google Scholar
Seki, M., Carninci, P., Nishiyama, Y., Hayashizaki, Y. & Shinozaki, K. High-efficiency cloning of Arabidopsis full-length cDNA by biotinylated CAP trapper. Plant J. 15, 707–720 (1998).
Article CAS PubMed Google Scholar
Seki, M. et al. Functional annotation of a full-length Arabidopsis cDNA collection. Science. 296, 141–145 (2002).
Article ADS PubMed Google Scholar
Berrow, N. S. et al. A versatile ligation-independent cloning method suitable for high-throughput expression screening applications. Nucleic Acids Res. 35, e45, doi:10.1093/nar/gkm047 (2007).
Article ADS PubMed PubMed Central Google Scholar
Hunter, S. et al. InterPro: the integrative protein signature database. Nucleic Acids Res. 37, D211–D215 (2009).
Article CAS PubMed Google Scholar
Saski, R. & Pizer, L. I. Regulatory properties of purified 3-phosphoglycerate dehydrogenase from Bacillus subtilis. Eur. J. Biochem. 51, 415–427 (1975).
Article CAS PubMed Google Scholar
Michaelis, L. & Menten, M. L. Die kinetik der invertinwirkung. Biochem. Z 49, 352 (1913).
Google Scholar

Download references

Acknowledgements

We are grateful to Drs. Shigeki Furuya (Kyushu University Bio-Architecture Center), Makoto Nishiyama (Biotechnology Research Center, The University of Tokyo) and Kansuporn Sriyudthsak (RIKEN Center for Sustainable Resource Science) for discussions and suggestions. This research was supported in part by CREST, JST to M.Y.H. and JSPS KAKENHI Grant Numbers 26870855 to E.O and 25113010 to M.Y.H.

Author information

Authors and Affiliations

RIKEN Center for Sustainable Resource Science, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan
Eiji Okamura & Masami Yokota Hirai

Authors

Eiji Okamura
View author publications
You can also search for this author in PubMed Google Scholar
Masami Yokota Hirai
View author publications
You can also search for this author in PubMed Google Scholar

Contributions

E.O. and M.Y.H. designed the research. E.O. performed all experiments. E.O. and M.Y.H. analyzed the data. E.O. and M.Y.H. wrote the manuscript.

Corresponding author

Correspondence to Masami Yokota Hirai.

Ethics declarations

Competing Interests

The authors declare that they have no competing interests.

Additional information

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Supplementary Information

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and permissions

About this article

Cite this article

Okamura, E., Hirai, M.Y. Novel regulatory mechanism of serine biosynthesis associated with 3-phosphoglycerate dehydrogenase in Arabidopsis thaliana . Sci Rep 7, 3533 (2017). https://doi.org/10.1038/s41598-017-03807-5

Download citation

Received: 21 December 2016
Accepted: 17 May 2017
Published: 14 June 2017
DOI: https://doi.org/10.1038/s41598-017-03807-5
Springer Nature Limited

This article is cited by

The phosphorylated pathway of serine biosynthesis affects sperm, embryo, and sporophyte development, and metabolism in Marchantia polymorpha
- Mengyao Wang
- Hiromitsu Tabeta
- Masami Yokota Hirai
Communications Biology (2024)
Transcriptomics Reveals the Mechanism of Purpureocillium lilacinum GZAC18-2JMP in Degrading Keratin Material
- Shumei Han
- Yingxia Lu
- Yanfeng Han
Current Microbiology (2024)
Bioenergetics of pollen tube growth in Arabidopsis thaliana revealed by ratiometric genetically encoded biosensors
- Jinhong Liu
- Shey-Li Lim
- Boon Leong Lim
Nature Communications (2022)
Seed photorespiration: a perspective review
- Marcia Eugenia Amaral Carvalho
- Carlos Alberto Labate
- Ricardo Antunes Azevedo
Plant Growth Regulation (2022)
Comparative proteomic analysis reveals that the Heterosis of two maize hybrids is related to enhancement of stress response and photosynthesis respectively
- Daoping Wang
- Yongying Mu
- Yinghong Pan
BMC Plant Biology (2021)

Novel regulatory mechanism of serine biosynthesis associated with 3-phosphoglycerate dehydrogenase in Arabidopsis thaliana

Abstract

Similar content being viewed by others

Introduction

Domain structures and biochemical properties of AtPGDHs

2-Oxoglutarate is not a good substrate of AtPGDHs

Novel cooperative activators of AtPGDHs

Domains required for regulation by effector amino acids

Combined effects of l-serine and amino acid activators

Discussion

Methods

Multiple alignments of amino acid sequences

Reagents

Plant materials

Preparation of recombinant enzymes

Estimation of molecular weights of recombinant AtPGDHs

Spectrophotometric assays of recombinant AtPGDHs

Non-linear regression with Hill equations

References

Acknowledgements

Author information

Authors and Affiliations

Contributions

Corresponding author

Ethics declarations

Competing Interests

Additional information

Electronic supplementary material

Rights and permissions

About this article

Cite this article

Share this article

This article is cited by

Search

Navigation