Recognition signal for the C‐terminal processing protease of D1 precursor protein in the photosystem II reaction center An analysis using synthetic oligopeptides

Abstract

Synthetic oligopeptides of different chain lengths of 11 to 38 amino acids, corresponding to the carboxyl-terminal sequence of D 1 precursor protein of the photosystem II reaction center, were subjected to a proteolytic cleavage by a processing enzyme isolated from spinach, in order to analyze the recognition signal. Practically the same Km and V~ x values were obtained for the oligopeptides consisting of more than 19 amino acids; a decrease in affinity, without affecting the Vma × value, was observed for the peptide consisting of 16 amino acids; no detectable activity was found for the peptide with 11 amino acids. When Asp-342 (12th residue from C-terminus) was replaced with Asn, for the peptide consisting of 16 amino acids, the enzymatic activity was completely abolished. In contrast, replacing Asp-342 with Glu had little effect. The efficiency of these oligopeptides as a substrate mentioned above, together with their effectiveness as an inhibitor, clearly demonstrated that the negative charge on Asp-342 plays a crucial role in the recognition, i.e. binding and cleavage, of the substrate by the processing enzyme, and suggested that the carboxyl-terminal extension consisting of 9 amino acids, by itself is not important in the binding.

The ctpA gene encodes the C-terminal processing protease for the D1 protein of the photosystem II reaction center complex.

“…It is noteworthy that three Arg residues in the CtpA protein (residues 223, 255, and 327) are completely conserved in all of the IRBPs. One of these residues in CtpA may form a salt bridge with the Asp-342 residue (of the D1 protein), the negative charge on which has recently been implicated in playing a critical role in binding of the C-terminal region of pD1 to the processing protease (37).…”

Membrane Lipid Unsaturation Modulates Processing of the Photosystem II Reaction-Center Protein D1 at Low Temperatures

“…The enzyme involved in the carboxyl-terminal processing of the pD1 protein has been isolated and partially purified from higher plants (Inagaki et al, 1989;Bowyer et al, 1992;Taguchi et al, 1993;Fujita et al, 1995) and from an alga, Scenedesmus obliquus (Taylor et al, 1988;Bowyer et al, 1992). Similarly, a nove1 gene (ctpA) recently isolated in Synechocystis sp.…”

“…The D1 protein is known to be synthesized on thylakoid-bound ribosomes (Minami and Watanabe, 1984;Herrin and Michaels, 1985) as a precursor form with a 9-to 16-amino acid carboxyl-terminal extension (Reisfeld et al, 1982;Marder et al, 1984;Takahashi et al, 1988). After insertion into the thylakoid membrane, pD1 is processed at its carboxyl-terminal end by a specific lumenal protease (Reisfeld et al, 1982;Taylor et al, 1988;Inagaki et al, 1989;Taguchi et al, 1993;Fujita et al, 1995) that cleaves the extension at amino acid 344, resulting in a mature D1 protein of equal size in both plants and cyanobacteria.…”

Molecular Studies of CtpA, the Carboxyl-terminal Processing Protease for the D1 Protein of the Photosystem II Reaction Center in Higher Plants

“…In most cases, the D1 subunit is synthesized as a precursor polypeptide with a short carboxyl-terminal extension (8,9). The C terminus of this protein is subsequently translocated into the thylakoid lumen and immediately cleaved off by a lumenal C-terminal processing peptidase (8,10). Mutants that are unable to remove this extension fail to produce oxygen, presumably because the C terminus of the mature D1 protein functions as a ligand for the manganese cluster in PSII (11)(12)(13).…”

“…The D1-processing protease has been partially purified from pea and spinach (10,14,15). Recently, ctpA, the gene encoding this protease, has been isolated from the cyanobacterium Synechocystis 6803 (16).…”