Mutations in the small subunit of cyanobacterial ribulose-bisphosphate carboxylase/oxygenase that modulate interactions with large subunits.
In the cyanobacterium Anacystis nidulans (Synechococcus PCC6301), ribulose 1,5-bisphosphate carboxylase/oxygenase (Rbu-P2 carboxylase) is composed of eight large subunits and eight small subunits. There are three regions of the small subunit that contain amino acids that are conserved throughout evolution, from bacteria to higher plants. Since the function of the small subunit is not fully understood, site-directed mutagenesis was performed on highly conserved residues in the first and second conserved regions. Ser-16, Pro-19, Leu-21, and Tyr-54 were replaced by Asp-16, His-19, Glu-21, and Ser-54, respectively. Crude extracts containing the recombinant His-19 mutant enzyme indicated that there was little effect on either Rbu-P2 carboxylase activity or interactions between large and small subunits. However, the Asp-16, Glu-21, and Ser-54 mutations showed effects on Rbu-P2 carboxylase activity and the interaction between large and small subunits. The large and small subunits of the Asp-16, Glu-21, and Ser-54 enzymes were found to dissociate during nondenaturing gel electrophoresis or sucrose density gradient centrifugation. However, the dissociated small subunits remained functional and were capable of reconstituting Rbu-P2 carboxylase activity when added to large subunits. These results indicated that Ser-16, Leu-21, and Tyr-54 might play an important role in interactions between large and small subunits of the A. nidulans enzyme.
Lee
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Department of Microbiology, Ohio State University, Columbus 43210.
Berka
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Tabita
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D000595:Amino Acid Sequence; D001483:Base Sequence; D000458:Cyanobacteria / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics* / Q000378:metabolism
D012273:Ribulose-Bisphosphate Carboxylase