Replacement of a conserved arginine in the assembly domain of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit interferes with holoenzyme formation.
In higher plants the small subunit (S) of ribulose-1,5-bisphosphate carboxylase/oxygenase (ribulose-P2 carboxylase, EC 4.1.1.39) contains a segment of 16 amino acids which is absent from cyanobacterial S. This segment connecting two beta sheets has been shown, by crystallographic analysis, to form a hairpin loop. The quaternary structure of ribulose-P2 carboxylase indicates several S to large subunit (L) interactions. Eleven of 22 residues within the loop form the interface with 20 residues from two different L dimers. Eight of the loop residues are involved in hydrogen bonds, salt links, and hydrophobic interactions. To test the hypothesis, whether this loop had a function in the assembly of L and S into the hexadecameric enzyme, 6 amino acids within the loop were modified by site-directed mutagenesis of the pea rbcS-3A gene. All substituted S were imported by isolated chloroplasts from pea with wild type efficiency. Mutants E54-R, H55-A, P59-A, D63-G, D63-L, and Y66-A were assembly-competent, indicating that changes of side chains at these positions are tolerated. Replacement of arginine 53, whose side chain forms H-bonds with L residues Y226 and G261, with glutamate completely abolished assembly into holoenzyme. We suggest that arginine 53 in S is essential for ribulose-P2 carboxylase quaternary structure in higher plants.
Flachmann
R
R
Department of Biochemistry, University of Arizona, Tucson 85721.
Bohnert
H J
HJ
D000595:Amino Acid Sequence; D001120:Arginine / Q000378:metabolism*; D001483:Base Sequence; D002736:Chloroplasts / Q000378:metabolism; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010944:Plants / Q000201:enzymology; D011487:Protein Conformation; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics* / Q000378:metabolism
D004247:DNA; D001120:Arginine; D012273:Ribulose-Bisphosphate Carboxylase