Substitutions at the Asp-473 latch residue of chlamydomonas ribulosebisphosphate carboxylase/oxygenase cause decreases in carboxylation efficiency and CO(2)/O(2) specificity.
The loop between alpha-helix 6 and beta-strand 6 in the alpha/beta-barrel active site of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) plays a key role in discriminating between gaseous substrates CO(2) and O(2). Based on numerous x-ray crystal structures, loop 6 is either closed or open depending on the presence or absence, respectively, of substrate ligands. The carboxyl terminus folds over loop 6 in the closed conformation, prompting speculation that it may trigger or latch loop 6 closure. Because an x-ray crystal structure of tobacco Rubisco revealed that phosphate is located at a site in the open form that is occupied by the carboxyl group of Asp-473 in the closed form, it was proposed that Asp-473 may serve as the latch that holds the carboxyl terminus over loop 6. To assess the essentiality of Asp-473 in catalysis, we used directed mutagenesis and chloroplast transformation of the green alga Chlamydomonas reinhardtii to create D473A and D473E mutant enzymes. The D473A and D473E mutant strains can grow photoautotrophically, indicating that Asp-473 is not essential for catalysis. However, both substitutions caused 87% decreases in carboxylation catalytic efficiency (V(max)/K(m)) and approximately 16% decreases in CO(2)/O(2) specificity. If the carboxyl terminus is required for stabilizing loop 6 in the closed conformation, there must be additional residues at the carboxyl terminus/loop 6 interface that contribute to this mechanism. Considering that substitutions at residue 473 can influence CO(2)/O(2) specificity, further study of interactions between loop 6 and the carboxyl terminus may provide clues for engineering an improved Rubisco.
Satagopan
Sriram
S
Department of Biochemistry, University of Nebraska, Lincoln, Nebraska 68588-0664, USA.
Spreitzer
Robert J
RJ
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics; D002245:Carbon Dioxide / Q000378:metabolism*; D002384:Catalysis; D016825:Chlamydomonas reinhardtii / Q000201:enzymology* / Q000235:genetics; D006358:Hot Temperature; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010100:Oxygen / Q000378:metabolism*; D010641:Phenotype; D017434:Protein Structure, Tertiary; D012273:Ribulose-Bisphosphate Carboxylase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013379:Substrate Specificity; D014170:Transformation, Genetic
D002245:Carbon Dioxide; D001224:Aspartic Acid; D012273:Ribulose-Bisphosphate Carboxylase; D010100:Oxygen