scholarly journals Biochemical characterization of phosphoenolpyruvate carboxykinases from Arabidopsis thaliana

2019 ◽  
Vol 476 (20) ◽  
pp. 2939-2952 ◽  
Author(s):  
Bruno E. Rojas ◽  
Matías D. Hartman ◽  
Carlos M. Figueroa ◽  
Laura Leaden ◽  
Florencio E. Podestá ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
High Throughput Screening ◽  
Co2 Fixation ◽  
Biochemical Characterization ◽  
Cam Plants ◽  
Recombinant Production ◽  
C3 Species ◽  
Regulatory Properties ◽  
Allosteric Regulators

Abstract ATP-dependent phosphoenolpyruvate carboxykinases (PEPCKs, EC 4.1.1.49) from C4 and CAM plants have been widely studied due to their crucial role in photosynthetic CO2 fixation. However, our knowledge on the structural, kinetic and regulatory properties of the enzymes from C3 species is still limited. In this work, we report the recombinant production and biochemical characterization of two PEPCKs identified in Arabidopsis thaliana: AthPEPCK1 and AthPEPCK2. We found that both enzymes exhibited high affinity for oxaloacetate and ATP, reinforcing their role as decarboxylases. We employed a high-throughput screening for putative allosteric regulators using differential scanning fluorometry and confirmed their effect on enzyme activity by performing enzyme kinetics. AthPEPCK1 and AthPEPCK2 are allosterically modulated by key intermediates of plant metabolism, namely succinate, fumarate, citrate and α-ketoglutarate. Interestingly, malate activated and glucose 6-phosphate inhibited AthPEPCK1 but had no effect on AthPEPCK2. Overall, our results demonstrate that the enzymes involved in the critical metabolic node constituted by phosphoenolpyruvate are targets of fine allosteric regulation.

scholarly journals Biochemical characterization of the novel endo -β-mannanase At Man5-2 from Arabidopsis thaliana

Plant Science ◽  
2015 ◽  
Vol 241 ◽  
pp. 151-163 ◽  
Author(s):  
Yang Wang ◽  
Shoaib Azhar ◽  
Rosaria Gandini ◽  
Christina Divne ◽  
Ines Ezcurra ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Biochemical Characterization ◽  
The Novel

scholarly journals Functional and biochemical characterization of a recombinant Arabidopsis thaliana 3-deoxy-D-manno-octulosonate 8-phosphate synthase

2004 ◽  
Vol 381 (1) ◽  
pp. 185-193 ◽  
Author(s):  
Jing WU ◽  
Mayur A. PATEL ◽  
Appavu K. SUNDARAM ◽  
Ronald W. WOODARD
Keyword(s):  
Arabidopsis Thaliana ◽  
Molecular Mass ◽  
Biochemical Characterization ◽  
Dipicolinic Acid ◽  
Kinetic Studies ◽  
Maximum Activity ◽  
Reading Frame ◽  
Sequential Mechanism ◽  
Phosphate Synthase

An open reading frame, encoding for KDOPS (3-deoxy-D-manno-octulosonate 8-phosphate synthase), from Arabidopsis thaliana was cloned into a T7-driven expression vector. The protein was overexpressed in Escherichia coli and purified to homogeneity. Recombinant A. thaliana KDOPS, in solution, displays an apparent molecular mass of 76 kDa and a subunit molecular mass of 31.519 kDa. Unlike previously studied bacterial KDOPSs, which are tetrameric, A. thaliana KDOPS appears to be a dimer in solution. The optimum temperature of the enzyme is 65 °C and the optimum pH is 7.5, with a broad peak between pH 6.5 and 9.5 showing 90% of maximum activity. The enzyme cannot be inactivated by EDTA or dipicolinic acid treatment, nor it can be activated by a series of bivalent metal ions, suggesting that it is a non-metallo-enzyme, as opposed to the initial prediction that it would be a metallo-enzyme. Kinetic studies showed that the enzyme follows a sequential mechanism with Km=3.6 μM for phosphoenolpyruvate and 3.8 μM for D-arabinose 5-phosphate and kcat=5.9 s−1 at 37 °C. On the basis of the characterization of A. thaliana KDOPS and phylogenetic analysis, plant KDOPSs may represent a new, distinct class of KDOPSs.

scholarly journals Identification and biochemical characterization of the fructokinase gene family in Arabidopsis thaliana

2017 ◽  
Vol 17 (1) ◽  
Author(s):  
John W. Riggs ◽  
Philip C. Cavales ◽  
Sonia M. Chapiro ◽  
Judy Callis
Keyword(s):  
Arabidopsis Thaliana ◽  
Gene Family ◽  
Biochemical Characterization

Biochemical Characterization of Cytokinin Oxidases/Dehydrogenases from Arabidopsis thaliana Expressed in Nicotiana tabacum L.

2007 ◽  
Vol 26 (3) ◽  
pp. 255-267 ◽  
Author(s):  
Petr Galuszka ◽  
Hana Popelková ◽  
Tomáš Werner ◽  
Jitka Frébortová ◽  
Hana Pospíšilová ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Nicotiana Tabacum ◽  
Biochemical Characterization ◽  
Nicotiana Tabacum L

Biochemical characterization of Arabidopsis thaliana starch branching enzyme 2.2 reveals an enzymatic positive cooperativity

Biochimie ◽  
2017 ◽  
Vol 140 ◽  
pp. 146-158 ◽  
Author(s):  
A. Wychowski ◽  
C. Bompard ◽  
F. Grimaud ◽  
G. Potocki-Véronèse ◽  
C. D'Hulst ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Biochemical Characterization ◽  
Branching Enzyme ◽  
Starch Branching Enzyme ◽  
Positive Cooperativity

scholarly journals Production and analysis of a mammalian septin hetero-octamer complex

2020 ◽  
Author(s):  
Barry T. DeRose ◽  
Robert S. Kelley ◽  
Roshni Ravi ◽  
Bashkim Kokona ◽  
Elias T. Spiliotis ◽  
...  
Keyword(s):  
Cell Biology ◽  
Biochemical Characterization ◽  
Fluorescent Protein ◽  
Mechanistic Study ◽  
Central Position ◽  
Protein Fusion ◽  
Recombinant Production ◽  
And Function ◽  
Order Structures

AbstractThe septins are filament-forming proteins found in diverse eukaryotes from fungi to vertebrates, with roles in cytokinesis, shaping of membranes and modifying cytoskeletal organization. These GTPases assemble into rod-shaped soluble hetero-hexamers and hetero-octamers in mammals, which polymerize into filaments and higher order structures. While the cell biology and pathobiology of septins are advancing rapidly, mechanistic study of the mammalian septins is limited by a lack of recombinant hetero-octamer materials. We describe here the production and characterization of a recombinant mammalian septin hetero-octamer of defined stoichiometry, the SEPT2/SEPT6/SEPT7/SEPT3 complex. Using a fluorescent protein fusion to the complex, we observed filaments assembled from this complex. In addition, we used this novel tool to resolve recent questions regarding the organization of the soluble septin complex. Biochemical characterization of a SEPT3 truncation that disrupts SEPT3-SEPT3 interactions is consistent with SEPT3 occupying a central position in the complex while the SEPT2 subunits are at the ends of the rod-shaped octameric complexes. Consistent with SEPT2 being on the complex ends, we find that our purified SEPT2/SEPT6/SEPT7/SEPT3 hetero-octamer copolymerizes into mixed filaments with separately purified SEPT2/SEPT6/SEPT7 hetero-hexamer. We expect this new recombinant production approach to lay essential groundwork for future studies into mammalian septin mechanism and function.

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