N-terminal truncation affects the kinetics and structure of fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase from Arabidopsis thaliana

VILLADSEN, Dorthe; Nielsen, Theodor

doi:10.1042/bj3590591

Cited by 14 publications

(7 citation statements)

References 27 publications

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“…The phosphosite of interest, Ser276, is within the plant-specific regulatory N-terminal domain (42,76) but is not among the phosphosites in the known 14-3-3 binding site (77). Ser276 is regulated by SnRK1 (46) and is conserved in many plant species (Figure S 8A).…”

Section: A Rhythmic F2kp Phosphosite Is Biochemically Relevantmentioning

confidence: 99%

The circadian clock gene circuit controls protein and phosphoprotein rhythms inArabidopsis thaliana

Krahmer

Hindle

Perby

et al. 2019

Preprint

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The 24-hour rhythmicity in the levels of many eukaryotic mRNAs contrasts with the long half-lives of most detected proteins. Such stable molecular species are not expected to reflect the RNA rhythms, emphasizing the need to test the circadian regulation of protein abundance and modification. Here we present circadian proteomic and comparable phosphoproteomic time-series from Arabidopsis thaliana plants, estimating that 0.4% of quantified proteins and a much larger proportion of quantified phosphosites were rhythmic under constant light conditions. Approximately half of the quantified phospho-sites were most phosphorylated at subjective dawn, when SnRK1 protein kinase was a candidate regulator.A CCA1-over-expressing line that disables the plant clock gene circuit lacked most or all circadian protein phosphorylation, indicating that the phospho-dawn is regulated by the canonical clock mechanism. The few phospho-sites that fluctuated despite CCA1-over-expression still tended to peak in abundance close to subjective dawn, indicating that their temporal regulation was less dependent on the clock gene circuit. To test the potential functional relevance of our datasets, we conduct phosphomimetic experiments using the bifunctional enzyme fructose-6-phosphate-2-kinase / phosphatase (F2KP), as an example. The clock gene circuit controls diverse protein targets in metabolism and physiology via phosphorylation, including where the bulk abundance of the cognate proteins is arrhythmic.

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Section: A Rhythmic F2kp Phosphosite Is Biochemically Relevantmentioning

confidence: 99%

The circadian clock gene circuit controls protein and phosphoprotein rhythms inArabidopsis thaliana

Krahmer

Hindle

Perby

et al. 2019

Preprint

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“…In addition, extensive studies of hydantoinases from various microorganisms such as strain screening, gene cloning, protein purification, dependence of metal ions, reaction mechanism, and three-dimensional conformation have been reported [6][7][8]. As one of the effective ways to study the relationship between protein structure and function, the site-directed mutagenesis and deletion mutation have been widely used [9,10]. Recently, Cheon et al [11] reported that some stereochemistry gate loops in the D-hydantoinase from B. stearothermophilus SD1 constituted a hydrophobic substrate binding pocket, and its mutants (Y155F, Y155E, L65E, and F159E) located at these loops might cause a dramatic decrease in enzymatic activity for two different substrates, non-substituted hydantoin and hydroxylphenyl-hydantoin (HPH).…”

Section: Introductionmentioning

confidence: 99%

The Flexibility of the Non-Conservative Region at the C Terminus of d-Hydantoinase from Pseudomonas putida YZ-26 is Extremely Limited

Zhang

Niu

Shi

et al. 2007

Appl Biochem Biotechnol

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We previously reported that a deletion mutant (P478) with a residue Arg deleted at the C terminus of D-hydantoinase (P479) from Pseudomonas putida YZ-26 was dissociated into the monomer from its dimeric state. Based on the above result, a series of mutants of the enzyme with the C-terminal residues either deleted or substituted were prepared. The size-exclusion chromatography and bioactivity assay show that a C-terminal-substituted enzyme (R479D) and several truncated mutants (P478, P477, P476, and P475) are dissociated into the monomeric state as well, but their activities are largely retained. In contrast, two other mutants (R474 and R479A) are expressed in the form of random aggregates without any activity. Our experiments demonstrate that only the last four amino acids (-PVQR) at the C terminus of the enzyme can be deleted without seriously affecting its activity, although the enzyme is dissociated from a dimer into a monomer. These mutants also reveal some unique properties such as the enzymatic activity in vivo or in vitro, the effect of divalent metal ions, and the thermostability etc. in comparison to wild-type enzyme (P479). In addition, the three-dimensional structural modeling shows that the intact structure of the enzyme is essential, and the flexibility of the non-conservative region at the C terminus of the enzyme is quite limited.

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“…Purification of the recombinant protein from Arabidopsis (AT1G07110) revealed that the kinase domain is inhibited by PPi, PEP, 3PGA, 2PGA and DHAP, whereas Pi and Pyr activate the kinase domain. On the other hand, the phosphatase domain is inhibited by Pi, Fru6P and FBP (Villadsen & Nielsen, 2001). Removal of the N-terminal extension led to an overall decrease in catalysis but did not affect the regulation by metabolites exhibited by the enzyme (Villadsen & Nielsen, 2001).…”

Section: The Conversion Of Pep Into Pyrmentioning

confidence: 95%

The involvement of allosteric effectors and post‐translational modifications in the control of plant central carbon metabolism

et al. 2023

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SUMMARY Plant metabolism is finely orchestrated to allow the occurrence of complementary and sometimes opposite metabolic pathways. In part this is achieved by the allosteric regulation of enzymes, which has been a cornerstone of plant research for many decades. The completion of the Arabidopsis genome and the development of the associated toolkits for Arabidopsis research moved the focus of many researchers to other fields. This is reflected by the increasing number of high‐throughput proteomic studies, mainly focused on post‐translational modifications. However, follow‐up ‘classical’ biochemical studies to assess the functions and upstream signaling pathways responsible for such modifications have been scarce. In this work, we review the basic concepts of allosteric regulation of enzymes involved in plant carbon metabolism, comprising photosynthesis and photorespiration, starch and sucrose synthesis, glycolysis and gluconeogenesis, the oxidative pentose phosphate pathway and the tricarboxylic acid cycle. Additionally, we revisit the latest results on the allosteric control of the enzymes involved in these pathways. To conclude, we elaborate on the current methods for studying protein–metabolite interactions, which we consider will become crucial for discoveries in the future.

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N-terminal truncation affects the kinetics and structure of fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase from Arabidopsis thaliana

Cited by 14 publications

References 27 publications

The circadian clock gene circuit controls protein and phosphoprotein rhythms inArabidopsis thaliana

The circadian clock gene circuit controls protein and phosphoprotein rhythms inArabidopsis thaliana

The Flexibility of the Non-Conservative Region at the C Terminus of d-Hydantoinase from Pseudomonas putida YZ-26 is Extremely Limited

The involvement of allosteric effectors and post‐translational modifications in the control of plant central carbon metabolism

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