Evolution of C4 phosphoenolpyruvate carboxylase in Flaveria, a conserved serine residue in the carboxyl-terminal part of the enzyme is a major determinant for C4-specific characteristics.
C4 phosphoenolpyruvate carboxylases have evolved from ancestral C3 isoforms during the evolution of angiosperms and gained distinct kinetic and regulatory properties compared with the C3 isozymes. To identify amino acid residues and/or domains responsible for these C4-specific properties the C4 phosphoenolpyruvate carboxylase of Flaveria trinervia (C4) was compared with its orthologue in the closely related C3 plant Flaveria pringlei. Reciprocal enzyme chimera were constructed and the kinetic constants, K(0.5) and k(cat), as well as the Hill coefficient, h, were determined for the substrate phosphoenolpyruvate both in the presence and absence of the activator glucose 6-phosphate. By this approach two regions were identified which determined most of the kinetic differences of the C4 and C3 ppcA phosphoenolpyruvate carboxylases with respect to the substrate PEP. In addition, the experiments suggest that the two regions do not act additively but interact with each other. The region between amino acids 296 and 437 is essential for activation by glucose 6-phosphate. The carboxyl-terminal segment between amino acids 645 and 966 contains a C4 conserved serine or a C3 invariant alanine at position 774 in the respective enzyme isoform. Site-directed mutagenesis shows that this position is a key determinant for the kinetic properties of the two isozymes.
Bläsing
O E
OE
Institut für Entwicklungs und Molekularbiologie der Pflanzen, Heinrich-Heine-Universität Düsseldorf, D-40225 Düsseldorf, Germany.
Westhoff
P
P
Svensson
P
P
D000409:Alanine; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D005075:Biological Evolution; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D019684:Magnoliopsida / Q000201:enzymology* / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010730:Phosphoenolpyruvate Carboxylase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010957:Plasmids; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012694:Serine; D013379:Substrate Specificity
D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D012694:Serine; D010730:Phosphoenolpyruvate Carboxylase; D000409:Alanine