A residue substitution in phosphoribulokinase of Synechocystis PCC 6803 renders the mutant light-sensitive.
We have isolated a light-sensitive mutant (BRLS) of the photosynthetic cyanobacterium Synechocystis 6803 (S. 6803) that does not survive exposure to bright light: 70% of BRLS cells die upon exposure to light of greater than 3,000 lux for 2 h. A complementing DNA fragment from wild-type cells and the corresponding DNA from the BRLS cells have been cloned and sequenced. An open reading frame is found to encode phosphoribulokinase, a key enzyme in the enzyme system for photosynthetic carbon reduction (ES-PCR). The deduced peptide sequence of this enzyme is highly homologous to eukaryotic phosphoribulokinases but is not similar to known prokaryotic phosphoribulokinases. The mutation responsible for the phenotype of BRLS is a single nucleotide change that results in substitution of phenylalanine for Ser-222 in the phosphoribulokinase. The catalytic activity and the apparent affinity for ATP of the mutated kinase are about one-tenth and one-seventh those of the wild-type kinase, respectively. Furthermore, the mutated kinase is selectively degraded in BRLS cells in bright light. Degradation of the mutated kinase and cell death in bright light can be suppressed by inhibiting photosynthetic electron flow (PS-EF) with 3-(3,4-dichlorophenyl)-1,1-dimethylurea. The data indicate that PS-EF is not impeded by an impaired ES-PCR although the ES-PCR activity is controlled by the rate of PS-EF. Continued PS-EF in the absence of the normal substrates for carbon reduction appears to result in damage to cellular components essential for life or in the generation of lethal components.
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Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138.
Bogorad
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D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D000458:Cyanobacteria / Q000201:enzymology* / Q000235:genetics; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D005822:Genetic Vectors; D007700:Kinetics; D008027:Light; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016366:Open Reading Frames; D010770:Phosphotransferases / Q000235:genetics* / Q000378:metabolism / Q000528:radiation effects; D017853:Phosphotransferases (Alcohol Group Acceptor); D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods; D011994:Recombinant Proteins / Q000378:metabolism / Q000528:radiation effects; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid
D011994:Recombinant Proteins; D004247:DNA; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C022390:phosphoribulokinase