Random mutagenesis targeted to the psbAII gene of Synechocystis sp. PCC 6803 to identify functionally important residues in the D1 protein of the photosystem II reaction center
Yamasato, A. (Okayama Univ. (Japan)) | Kamada, T. | Satoh, K.
More than one hundred mutants of Synechocystis sp. PCC 6803 impaired in photoautotrophic growth were generated by in vitro random PCR mutagenesis targeted to a region of the psbA2 gene corresponding to a 210 amino acid (Serl48-Ala357) segment of the D1 protein. The 90 random mutants that could translate the full-length D1 protein carried 1-9 (on average 3.0) amino acid substitutions in the targeted region. Mutations were often found in the obligate photoheterotrophic strains at specific residues that have been reported or speculated to be important in the function of PS2, such as Y161, H198, H272, E333 and H337. This verifies the usefulness of the present method to identify functionally important residues in PS2. Other residues that were often mutated in the strains with impaired photoautotrophy included non-charged residues around the lumenal edges of transmembrane helices C, D and E, such as I192 and N296. Eleven mutants carried a single-point mutation in residues, such as Q165, Q187, W278, A294 and N298, and these identified the functional importance of these residues, most of which were on the donor side of PS2. A preliminary characterization of some of the mutants obtained in this study is provided.
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