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Phosphorylation of bacterial-type phosphoenolpyruvate carboxylase at Ser425 provides a further tier of enzyme control in developing castor oil seeds
- Source :
- Biochemical Journal
- Publication Year :
- 2010
- Publisher :
- Portland Press Ltd., 2010.
-
Abstract
- PEPC [PEP (phosphoenolpyruvate) carboxylase] is a tightly controlled anaplerotic enzyme situated at a pivotal branch point of plant carbohydrate metabolism. Two distinct oligomeric PEPC classes were discovered in developing COS (castor oil seeds). Class-1 PEPC is a typical homotetramer of 107 kDa PTPC (plant-type PEPC) subunits, whereas the novel 910-kDa Class-2 PEPC hetero-octamer arises from a tight interaction between Class-1 PEPC and 118 kDa BTPC (bacterial-type PEPC) subunits. Mass spectrometric analysis of immunopurified COS BTPC indicated that it is subject to in vivo proline-directed phosphorylation at Ser425. We show that immunoblots probed with phosphorylation site-specific antibodies demonstrated that Ser425 phosphorylation is promoted during COS development, becoming maximal at stage IX (maturation phase) or in response to depodding. Kinetic analyses of a recombinant, chimaeric Class-2 PEPC containing phosphomimetic BTPC mutant subunits (S425D) indicated that Ser425 phosphorylation results in significant BTPC inhibition by: (i) increasing its Km(PEP) 3-fold, (ii) reducing its I50 (L-malate and L-aspartate) values by 4.5- and 2.5-fold respectively, while (iii) decreasing its activity within the physiological pH range. The developmental pattern and kinetic influence of Ser425 BTPC phosphorylation is very distinct from the in vivo phosphorylation/activation of COS Class-1 PEPC's PTPC subunits at Ser11. Collectively, the results establish that BTPC's phospho-Ser425 content depends upon COS developmental and physiological status and that Ser425 phosphorylation attenuates the catalytic activity of BTPC subunits within a Class-2 PEPC complex. To the best of our knowledge, this study provides the first evidence for protein phosphorylation as a mechanism for the in vivo control of vascular plant BTPC activity.
- Subjects :
- BTPC, bacterial-type phosphoenolpyruvate carboxylase
0106 biological sciences
PP2A, protein phosphatase type-2A
Mutant
Biology
oil seed metabolism
01 natural sciences
Biochemistry
PP2Ac, catalytic subunit of PP2A
03 medical and health sciences
COS, castor (Ricinus communis) oil seed(s)
Serine
Protein phosphorylation
AtPPC, plant-type phosphoenolpyruvate carboxylase isozyme from Arabidopsis thaliana
Phosphorylation
Molecular Biology
Ricinus communis (castor oil plant)
PTPC, plant-type PEPC
Plant Proteins
030304 developmental biology
2. Zero hunger
0303 health sciences
Kinase
PEP, phosphoenolpyruvate
Cell Biology
phosphorylation site-specific antibodies
Castor Bean
I50, inhibitor concentration producing 50% inhibition of enzyme activity
PEPC, PEP carboxylase
Phosphoenolpyruvate Carboxylase
protein phosphorylation
Pyruvate carboxylase
Kinetics
phosphoenolpyruvate carboxylase (PEPC)
Seeds
site-directed mutagenesis
Phosphoenolpyruvate carboxykinase
Phosphoenolpyruvate carboxylase
Protein Processing, Post-Translational
Research Article
RcPPC, BTPC from Ricinus communis
010606 plant biology & botany
Homotetramer
Subjects
Details
- ISSN :
- 14708728 and 02646021
- Volume :
- 433
- Database :
- OpenAIRE
- Journal :
- Biochemical Journal
- Accession number :
- edsair.doi.dedup.....2558f97d7d3163c4ef02af1f87807077
- Full Text :
- https://doi.org/10.1042/bj20101361