The Thiamin-Requiring 3 Mutation of Arabidopsis 5-Deoxyxylulose-Phosphate Synthase 1 Highlights How the Thiamin Economy Impacts the Methylerythritol 4-Phosphate Pathway

The thiamin-requiring mutants of Arabidopsis have a storied history as a foundational model for biochemical genetics in plants and have illuminated the central role of thiamin in metabolism. Recent integrative genetic and biochemical analyses of thiamin biosynthesis and utilization imply that leaf metabolism normally operates close to thiamin-limiting conditions. Thus, the mechanisms that allocate thiamin-diphosphate (ThDP) cofactor among the diverse thiamin-dependent enzymes localized in plastids, mitochondria, peroxisomes, and the cytosol comprise an intricate thiamin economy. Here, we show that the classical thiamin-requiring 3 (th3) mutant is a point mutation in plastid localized 5-deoxyxylulose synthase 1 (DXS1), a key regulated enzyme in the methylerythritol 4-phosphate (MEP) isoprene biosynthesis pathway. Substitution of a lysine for a highly conserved glutamate residue (E323) located at the subunit interface of the homodimeric enzyme conditions a hypomorphic phenotype that can be rescued by supplying low concentrations of thiamin in the medium. Analysis of leaf thiamin vitamers showed that supplementing the medium with thiamin increased total ThDP content in both wild type and th3 mutant plants, supporting a hypothesis that the mutant DXS1 enzyme has a reduced affinity for the ThDP cofactor. An unexpected upregulation of a suite of biotic-stress-response genes associated with accumulation of downstream MEP intermediate MEcPP suggests that th3 causes mis-regulation of DXS1 activity in thiamin-supplemented plants. Overall, these results highlight that the central role of ThDP availability in regulation of DXS1 activity and flux through the MEP pathway.

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Fast NMDA Receptor–Mediated Synaptic Currents in Neurons From Mice Lacking the ε2 (NR2B) Subunit

Journal of Neurophysiology ◽

10.1152/jn.2000.83.1.616 ◽

2000 ◽

Vol 83 (1) ◽

pp. 616-620 ◽

Cited By ~ 42

Author(s):

Kenneth R. Tovar ◽

Kathleen Sprouffske ◽

Gary L. Westbrook

Keyword(s):

Nmda Receptor ◽

Hippocampal Neurons ◽

Postsynaptic Membrane ◽

Synaptic Activity ◽

Wild Type ◽

Nmda Receptor Subunit ◽

Deactivation Kinetics ◽

Low Concentrations ◽

Specific Antagonist

The N-methyl-d-aspartate (NMDA) receptor has been implicated in the formation of synaptic connections. To investigate the role of the ε2 (NR2B) NMDA receptor subunit, which is prominently expressed during early development, we used neurons from mice lacking this subunit. Although ε2−/− mice die soon after birth, we examined whether NMDA receptor targeting to the postsynaptic membrane was dependent on the ε2 subunit by rescuing hippocampal neurons from these mice and studying them in autaptic cultures. In voltage-clamp recordings, excitatory postsynaptic currents (EPSCs) from ε2−/− neurons expressed an NMDA receptor–mediated EPSC that was apparent as soon as synaptic activity developed. However, compared with wild-type neurons, NMDA receptor–mediated EPSC deactivation kinetics were much faster and were less sensitive to glycine, but were blocked by Mg2+ or AP5. Whole cell currents from ε2−/− neurons were also more sensitive to block by low concentrations of Zn2+ and much less sensitive to the ε2-specific antagonist ifenprodil than wild-type currents. The rapid NMDA receptor–mediated EPSC deactivation kinetics and the pharmacological profile from ε2−/−neurons are consistent with the expression of ζ1/ε1 diheteromeric receptors in excitatory hippocampal neurons from mice lacking the ε2 subunit. Thus ε1 can substitute for the ε2 subunit at synapses and ε2 is not required for targeting of NMDA receptors to the postsynaptic membrane.

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THE ROLE OF TRANSCRIPTION FACTORS DFOXO, DSIR2 AND HSP70 IN LIFESPAN ALTERATION OF DROSOPHILA MELANOGASTER IN DIFFERENT LIGHT CONDITIONS

Ecological genetics ◽

10.17816/ecogen8367-80 ◽

2010 ◽

Vol 8 (3) ◽

pp. 67-80 ◽

Cited By ~ 2

Author(s):

Aleksey A Moskalev ◽

Olga A Malysheva

Keyword(s):

Drosophila Melanogaster ◽

Transcription Factors ◽

Stress Response ◽

Life Span ◽

Light Regime ◽

Light Conditions ◽

Wild Type ◽

Stress Response Genes ◽

Significant Difference

It was investigated the role of stress-response genes (dFOXO, dSir2, Hsp70) in regulation of life span of Drosophila in response to light regime alteration. It was revealed the FOXO-dependant mechanism of lifespan increasing at darkness conditions. The distance of lifespan of FOXO homozygous mutants at different light conditions were absent 3 times from 4 times. It was shown, that homozygotes with deletion of dSir2 have more significant difference between lifespan at standard light and darkness conditions with comparing to wild type and heterozygous strain. The same tendency was also detected the in the strains with Hsp70 deletions. It was produced the evidences of two mechanisms of light regime influence on lifespan: metabolism intensification at light conditions and neuroendocrine-determinated lifespan increasing at darkness conditions.

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Hsp90 Is Required for Pheromone Signaling in Yeast

Molecular Biology of the Cell ◽

10.1091/mbc.9.11.3071 ◽

1998 ◽

Vol 9 (11) ◽

pp. 3071-3083 ◽

Cited By ~ 86

Author(s):

Jean-François Louvion ◽

Toufik Abbas-Terki ◽

Didier Picard

Keyword(s):

Budding Yeast ◽

Heat Shock Protein 90 ◽

Wild Type ◽

Pheromone Signaling ◽

Endogenous Substrate ◽

Low Levels ◽

Biochemical Analyses ◽

Map Kinase Pathways

The heat-shock protein 90 (Hsp90) is a cytosolic molecular chaperone that is highly abundant even at normal temperature. Specific functions for Hsp90 have been proposed based on the characterization of its interactions with certain transcription factors and kinases including Raf in vertebrates and flies. We therefore decided to address the role of Hsp90 for MAP kinase pathways in the budding yeast, an organism amenable to both genetic and biochemical analyses. We found that both basal and induced activities of the pheromone-signaling pathway depend on Hsp90. Signaling is defective in strains expressing low levels or point mutants of yeast Hsp90 (Hsp82), or human Hsp90β instead of the wild-type protein. Ste11, a yeast equivalent of Raf, forms complexes with wild-type Hsp90 and depends on Hsp90 function for accumulation. For budding yeast, Ste11 represents the first identified endogenous “substrate” of Hsp90. Moreover, Hsp90 functions in steroid receptor and pheromone signaling can be genetically separated as the Hsp82 point mutant T525I and the human Hsp90β are specifically defective for the former and the latter, respectively. These findings further corroborate the view that molecular chaperones must also be considered as transient or stable components of signal transduction pathways.

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Critical role of Arg433 in rat transketolase activity as probed by site-directed mutagenesis

Biochemical Journal ◽

10.1042/bj3330367 ◽

1998 ◽

Vol 333 (2) ◽

pp. 367-372 ◽

Cited By ~ 17

Author(s):

Yunjo SOH ◽

Byoung J. SONG ◽

Jiingjau JENG ◽

Abraham T. KALLARAKAL

Keyword(s):

Critical Role ◽

Immunoblot Analysis ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Wild Type ◽

Hydrogen Bond Interactions ◽

Apparent Homogeneity ◽

Arginine Residues ◽

Biochemical Analyses

It has been shown that one arginine per monomer at an unknown position is essential for enzyme activity of the homodimeric transketolase (TK) [Kremer, Egan and Sable (1980) J. Biol. Chem. 255, 2405–2410]. To identify the critical arginine, four highly conserved arginine residues of rat TK (Arg102, Arg350, Arg433 and Arg506) were replaced with alanine by site-directed mutagenesis. Wild-type and mutant TK proteins were produced in Escherichia coli and characterized. The Arg102 → Ala mutant exhibited similar catalytic activity to the wild-type enzyme, whereas Arg350 → Ala, Arg506 → Ala and Arg433 → Ala mutants exhibited 36.7, 37.0 and 6.1% of the wild-type activity respectively. Three recombinant proteins (wild-type, Arg350 → Ala and Arg433 → Ala) were purified to apparent homogeneity using Ni2+-affinity chromatography and further characterized. All these proteins were able to form homodimers (148 kDa), as shown by immunoblot analysis subsequent to non-denaturing gel electrophoresis. The Arg433 → Ala mutant protein was less stable than the wild-type and Arg350 → Ala proteins at 55 °C. Kinetic analyses revealed that both Vmax and Km values were markedly affected in the Arg433 → Ala mutant. The Km values for two substrates xylulose 5-phosphate and ribose 5-phosphate were 11.5- and 24.3-fold higher respectively. The kcat/Km values of the Arg433 → Ala mutant for the two substrates were less than 1% of those of the wild-type protein. Molecular modelling of the rat TK revealed that Arg433 of one monomer has three potential hydrogen-bond interactions with the catalytically important highly conserved loop of the other monomer. Thus, our biochemical analyses and modelling data suggest the critical role of the previously uncharacterized Arg433 in TK activity.

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A strictly monofunctional bacterial hydroxymethylpyrimidine phosphate kinase precludes damaging errors in thiamin biosynthesis

Biochemical Journal ◽

10.1042/bcj20170437 ◽

2017 ◽

Vol 474 (16) ◽

pp. 2887-2895 ◽

Cited By ~ 5

Author(s):

Antje M. Thamm ◽

Gengnan Li ◽

Marlene Taja-Moreno ◽

Svetlana Y. Gerdes ◽

Valérie de Crécy-Lagard ◽

...

Keyword(s):

Comparative Analysis ◽

Kinase Activity ◽

Protein Family ◽

Biosynthesis Pathway ◽

Bacterial Genomes ◽

Thiamin Biosynthesis ◽

Biochemical Analyses ◽

The Cost

The canonical kinase (ThiD) that converts the thiamin biosynthesis intermediate hydroxymethylpyrimidine (HMP) monophosphate into the diphosphate can also very efficiently convert free HMP into the monophosphate in prokaryotes, plants, and fungi. This HMP kinase activity enables salvage of HMP, but it is not substrate-specific and so allows toxic HMP analogs and damage products to infiltrate the thiamin biosynthesis pathway. Comparative analysis of bacterial genomes uncovered a gene, thiD2, that is often fused to the thiamin synthesis gene thiE and could potentially encode a replacement for ThiD. Standalone ThiD2 proteins and ThiD2 fusion domains are small (∼130 residues) and do not belong to any previously known protein family. Genetic and biochemical analyses showed that representative standalone and fused ThiD2 proteins catalyze phosphorylation of HMP monophosphate, but not of HMP or its toxic analogs and damage products such as bacimethrin and 5-(hydroxymethyl)-2-methylpyrimidin-4-ol. As strictly monofunctional HMP monophosphate kinases, ThiD2 proteins eliminate a potentially fatal vulnerability of canonical ThiD, at the cost of the ability to reclaim HMP formed by thiamin turnover.

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Optimal Distribution Of Iron To Sink Organs Via Autophagy Is Important For Tolerance To Excess Zinc In Arabidopsis

Plant and Cell Physiology ◽

10.1093/pcp/pcab017 ◽

2021 ◽

Author(s):

Daiki Shinozaki ◽

Keitaro Tanoi ◽

Kohki Yoshimoto

Keyword(s):

Stress Responses ◽

Heavy Metal Stress ◽

Wild Type ◽

Major Pathway ◽

Growth Defects ◽

Intracellular Degradation ◽

The Media ◽

Healthy Growth ◽

Biochemical Analyses

Abstract Zinc (Zn) is a nutritionally essential metal element, but excess Zn in the environment is toxic to plants. Autophagy is a major pathway responsible for intracellular degradation. Here, we demonstrate the important role of autophagy in adaptation to excess Zn stress. We found that autophagy-defective Arabidopsis thaliana (atg2 and atg5) exhibited marked excess Zn–induced chlorosis and growth defects relative to wild-type. Imaging and biochemical analyses revealed that autophagic activity was elevated under excess Zn. Interestingly, the excess Zn symptoms of atg5 were alleviated by supplementation of high levels of iron (Fe) to the media. Under excess Zn, in atg5, Fe starvation was especially severe in juvenile true leaves. Consistent with this, accumulation levels of Fe3+ near the shoot apical meristem was remarkably reduced in atg5. Furthermore, excision of cotyledons induced severe excess Zn symptoms in wild-type, similar to those observed in atg5. Our data suggest that Fe3+ supplied from source leaves (cotyledons) via autophagy is distributed to sink leaves (true leaves) to promote healthy growth under excess Zn, revealing a new dimension, the importance of heavy-metal stress responses by the intracellular recycling.

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The Critical Role of Polyketide Synthase Gene On The Swainsonine Biosynthesis in The Fungus Metarhizium Anisopliae

10.21203/rs.3.rs-1092840/v1 ◽

2021 ◽

Author(s):

Lu Sun ◽

Enxia Huang ◽

Yu Zhang ◽

Ziyu Guo ◽

Kexin Wu ◽

...

Keyword(s):

Homologous Recombination ◽

Metarhizium Anisopliae ◽

Type Strain ◽

Polyketide Synthase ◽

Wild Type Strain ◽

Biosynthesis Pathway ◽

Wild Type ◽

Knock Out ◽

Polyketide Synthase Gene

Abstract Swainsonine (SW) is the principal toxic ingredient of locoweeds, and is produced by fungi including Metarhizium anisopliae, Slafractonia leguminicola, and Alternaria oxytropis. While the SW biosynthesis pathway of fungi and the catalytic enzyme genes that regulate synthesis are not cleanly. In this study, we used homologous recombination (HR) to knock out and interfere with the polyketide synthase gene (pks) of M. anisopliae to determine its effect on the SW biosynthesis pathway. The concentration of SW was measured in the fermentation broth of M. anisopliae at 1 d, 2 d, 3 d, 4 d, 5 d, 6 d or 7 d using LC-MS. The gene for the pks gene was detected by RT-qPCR. Day 5 of M. anisopliae gave the highest content of SW and the highest expression of the pks gene. To determine the role of the pks gene in the SW biosynthesis pathway of M. anisopliae, we used PEG-mediated homologous recombination (HR) to transform a wild-type strain (WT) with a Benomyl (ben)-resistant fragment to knock out the pks gene producing a mutant-type strain (MT) and used PEG-mediated RNAi to transform a wild-type strain (WT) with a Benomyl (ben)-resistant plasmid to interfere with the pks gene. A complemented-type (CT) strain was produced by adding a complementation vector that contains the geneticin (G418) resistance gene as a marker. The content of SW didn’t detected in MT strain, and returned to the original level in the CT strain, while the content of SW was significantly decreased in RNAi strain. We suggest that mutation and RNAi in the pks gene affect the cell wall formation of M. anisopliae, while the colony diameters, phenotypes, and growth rates did not change significantly, and no obvious changes in other cellular organelles were noted. These results indicate that the pks gene plays a crucial role in the SW biosynthesis of M. anisopliae, which provides an important theoretical basis for illuminating the SW biosynthesis and solving locoism in livestock.

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A cross-kingdom Nudix enzyme that pre-empts damage in thiamin metabolism

Biochemical Journal ◽

10.1042/bj20130516 ◽

2013 ◽

Vol 454 (3) ◽

pp. 533-542 ◽

Cited By ~ 26

Author(s):

Aymeric Goyer ◽

Ghulam Hasnain ◽

Océane Frelin ◽

Maria A. Ralat ◽

Jesse F. Gregory ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Phosphatase Activity ◽

Degradation Products ◽

Genomic Analysis ◽

Comparative Genomic Analysis ◽

Comparative Genomic ◽

Biosynthesis Pathway ◽

Free Standing ◽

Thiamin Diphosphate ◽

Thiamin Biosynthesis

Genes specifying the thiamin monophosphate phosphatase and adenylated thiazole diphosphatase steps in fungal and plant thiamin biosynthesis remain unknown, as do genes for ThDP (thiamin diphosphate) hydrolysis in thiamin metabolism. A distinctive Nudix domain fused to Tnr3 (thiamin diphosphokinase) in Schizosaccharomyces pombe was evaluated as a candidate for these functions. Comparative genomic analysis predicted a role in thiamin metabolism, not biosynthesis, because free-standing homologues of this Nudix domain occur not only in fungi and plants, but also in proteobacteria (whose thiamin biosynthesis pathway has no adenylated thiazole or thiamin monophosphate hydrolysis steps) and animals (which do not make thiamin). Supporting this prediction, recombinant Tnr3 and its Saccharomyces cerevisiae, Arabidopsis and maize Nudix homologues lacked thiamin monophosphate phosphatase activity, but were active against ThDP, and up to 60-fold more active against diphosphates of the toxic thiamin degradation products oxy- and oxo-thiamin. Deleting the S. cerevisiae Nudix gene (YJR142W) lowered oxythiamin resistance, overexpressing it raised resistance, and expressing its plant or bacterial counterparts restored resistance to the YJR142W deletant. By converting the diphosphates of damaged forms of thiamin into monophosphates, the Tnr3 Nudix domain and its homologues can pre-empt the misincorporation of damaged diphosphates into ThDP-dependent enzymes, and the resulting toxicity.

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Greater reductions in fat preferences in CALHM1 than CD36 knockout mice

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00015.2018 ◽

2018 ◽

Vol 315 (3) ◽

pp. R576-R585 ◽

Cited By ~ 5

Author(s):

Anthony Sclafani ◽

Karen Ackroff

Keyword(s):

Fatty Acid ◽

Knockout Mice ◽

Dietary Fat ◽

Taste Receptor ◽

Wild Type ◽

Short Term ◽

Low Concentrations ◽

Oil Emulsions ◽

Fat Preference

Several studies indicate an important role of gustation in intake and preference for dietary fat. The present study compared fat preference deficits produced by deletion of CD36, a putative fatty acid taste receptor, and CALHM1, an ion channel responsible for release of the ATP neurotransmitter used by taste cells. Naïve CD36 knockout (KO) mice displayed reduced preferences for soybean oil emulsions (Intralipid) at low concentrations (0.1–1%) compared with wild-type (WT) mice in 24 h/day two-bottle tests. CALHM1 KO mice displayed even greater Intralipid preference deficits compared with WT and CD36 KO mice. These findings indicate that there may be another taste receptor besides CD36 that contributes to fat detection and preference. After experience with concentrated fat (2.5–5%), CD36 KO and CALHM1 KO mice displayed normal preferences for 0.1–5% fat, although they still consumed less fat than WT mice. The experience-induced rescue of fat preferences in KO mice can be attributed to postoral fat conditioning. Short-term (3-min) two-bottle tests further documented the fat preference deficits in CALHM1 KO mice but also revealed residual preferences for concentrated fat (5–10%), which may be mediated by odor and/or texture cues.

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Epstein-Barr Virus Nuclear Protein EBNA3A Is Critical for Maintaining Lymphoblastoid Cell Line Growth

Journal of Virology ◽

10.1128/jvi.77.19.10437-10447.2003 ◽

2003 ◽

Vol 77 (19) ◽

pp. 10437-10447 ◽

Cited By ~ 46

Author(s):

Seiji Maruo ◽

Eric Johannsen ◽

Diego Illanes ◽

Andrew Cooper ◽

Elliott Kieff

Keyword(s):

Epstein Barr Virus ◽

Critical Role ◽

Growth Arrest ◽

Nuclear Antigen ◽

Wild Type ◽

Barr Virus ◽

Fusion Protein Expression ◽

Epstein Barr ◽

Biochemical Analyses

ABSTRACT To evaluate the role of Epstein-Barr Virus (EBV) nuclear antigen 3A (EBNA3A) in the continuous proliferation of EBV-infected primary B lymphocytes as lymphoblastoid cell lines (LCLs), we derived LCLs that are infected with a recombinant EBV genome that expresses EBNA3A fused to a 4-hydroxy-tamoxifen (4HT)-dependent mutant estrogen receptor hormone binding domain (EBNA3AHT). The LCLs grew similarly to wild-type LCLs in medium with 4HT despite a reduced level of EBNA3AHT fusion protein expression. In the absence of 4HT, EBNA3AHT moved from the nucleus to the cytoplasm and was degraded. EBNA3AHT-infected LCLs were unable to grow in medium without 4HT. The precise time to growth arrest varied inversely with cell density. Continued maintenance in medium without 4HT resulted in cell death, whereas readdition of 4HT restored cell growth. Expression of other EBNAs and LMP1, of CD23, and of c-myc was unaffected by EBNA3A inactivation. Wild-type EBNA3A expression from an oriP plasmid transfected into the LCLs protected the EBNA3AHT-infected LCLs from growth arrest and death in medium without 4HT, whereas EBNA3B or EBNA3C expression was unable to protect the LCLs from growth arrest and death. These experiments indicate that EBNA3A has a unique and critical role for the maintenance of LCL growth and ultimately survival. The EBNA3AHT-infected LCLs are also useful for genetic and biochemical analyses of the role of EBNA3A domains in LCL growth.

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