Molecular and structural analysis of C4-specific PEPC isoform from Pennisetum glaucum plays a role in stress adaptation.
Phosphoenolpyruvate carboxylase is an ubiquitous cytosolic enzyme that catalyzes the ß-carboxylation of phosphoenolpyruvate (PEP) and is encoded by multigene family in plants. It plays an important role in carbon economy of plants by assimilating CO2 into organic acids for subsequent C4 or CAM photosynthesis or to perform several anaplerotic roles in non-photosynthetic tissues. In this study, a cDNA clone encoding for PEPC polypeptide possessing signature motifs characteristic to ZmC4PEPC was isolated from Pennisetum glaucum (PgPEPC). Deduced amino acid sequence revealed its predicted secondary structure consisting of forty alpha helices and eight beta strands is well conserved among other PEPC homologs irrespective of variation in their primary amino acid sequences. Predicted PgPEPC quartenary structure is a tetramer consisting of a dimer of dimers,which is globally akin to maize PEPC crystal structure with respect to major chain folding wherein catalytically important amino acid residues of active site geometry are conserved. Recombinant PgPEPC protein expressed in E. coli and purified to homogeneity, possessed in vitro ß-carboxylation activity that is determined using a coupled reaction converting PEP into malate. Tetramer is the most active form, however, it exists in various oligomeric forms depending upon the protein concentration, pH, ionic strength of the media and presence of its substrate or effecters. Recombinant PgPEPC protein confers enhanced growth advantage to E. coli under harsh growth conditions in comparison to their respective controls; suggesting that PgPEPC plays a significant role in stress adaptation.
International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, 110067, India.
Jitender
J
Singh
Guda Maheedhara
GM
Reddy
Aakrati
A
Agarwal
Kottakota
K
Chandrasekhar
S K
SK
Sopory
M K
MK
Reddy
Tanushri
T
Kaul
D000222:Adaptation, Physiological / Q000235:genetics*; D000595:Amino Acid Sequence; D020134:Catalytic Domain; D015870:Gene Expression; D015723:Gene Library; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D031782:Pennisetum / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D010730:Phosphoenolpyruvate Carboxylase / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010802:Phylogeny; D018515:Plant Leaves / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020033:Protein Isoforms; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D010940:Plant Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D010730:Phosphoenolpyruvate Carboxylase