Abstract
CtpA, a carboxyl-terminal processing protease, is a member of a novel family of endoproteases that includes a tail-specific protease from Escherichia coli. In oxygenic photosynthetic organisms, CtpA catalyzes C-terminal processing of the D1 protein of photosystem II, an essential event for the assembly of a manganese cluster and consequent light-mediated water oxidation. We introduced site-specific mutations at 14 conserved residues of CtpA in the cyanobacterium Synechocystis sp. PCC 6803 to examine their functional roles. Analysis of the photoautotrophic growth capabilities of these mutants, their ability to process precursor D1 protein and hence evolve oxygen, along with an estimation of the protease content in the mutants revealed that five of these residues are critical for in vivo activity of CtpA. Recent x-ray crystal structure analysis of CtpA from the eukaryotic alga Scenedesmus obliquus (Liao, D.-I., Qian, J., Chisholm, D. A., Jordan, D. B. and Diner, B. A. (2000) Nat. Struct. Biol. 7, 749-753) has shown that the residues equivalent to Ser-313 and Lys-338, two of the five residues mentioned above, form the catalytic center of this enzyme. Our in vivo analysis demonstrates that the three other residues, Asp-253, Arg-255, and Glu-316, are also important determinants of the catalytic activity of CtpA.
Highlights
During recent years, a new class of endoproteases with carboxyl-terminal processing activities has been described in various bacteria and organellar systems [1,2,3]
The E316D mutant could grow photoautotrophically, whereas the E316Q mutant could not. The latter mutant strain did not exhibit any photosystem II (PSII) activity and accumulated only the precursor form of D1 (Fig. 5). These results indicated that the presence of a negatively charged residue at position 316 is essential for the catalytic activity of Results shown are those from a representative experiment
The high specific activity of this endoprotease is a critical determinant during the assembly of functionally competent PSII complexes
Summary
Materials—All chemicals used were of reagent grade. Enzymes for recombinant DNA work were from New England Biolabs (Beverly, MA). Bacterial Strains and Culture Conditions—Synechocystis 6803 wild type and mutant cells were grown at 30 °C under 50 mol of photons mϪ2 sϪ1 of white light in BG11 medium [20]. A ⌬ctpA mutant was used as the background strain to generate various site-specific mutations in the ctpA gene. The ⌬ctpA mutant strain was generated by transforming wild type Synechocystis 6803 cells with the recombinant plasmid pSL795. The CtpAk strain was used as a positive control in the current study It was generated by transforming the ⌬ctpA mutant with the pSL958 plasmid (Fig. 1). The generation of the desired mutants was achieved by transforming ⌬ctpA cells with individual plasmids using a previously described procedure [23, 24]. The half-life of the precursor form of the D1 protein in each mutant was estimated by analyzing the resultant fluorographs using the Intelligent Quantifier software (BioImage)
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