Genetic Engineering of Herbicide Resistance: Saturation Mutagenesis of Isoleucine 229 of the Reaction Center L Subunit

Abstract

Oligonucleotide-mediated mutagenesis has been used to change isoleucine 229 of the reaction center L subunit of Rhodopseudomonas capsulata to seventeen other amino acids. A system of deletion strains and complementing plasmids has been used to assay the effects of changing this residue, which forms part of the secondary quinone Oв binding site. The reaction center is assembled in all seventeen mutants. Ten of the seventeen mutations (Gly, His, Tyr, Trp, Asn, Asp. Gin, Glu, Arg, and Lys) result in the loss of photosynthetic growth. The remaining seven mutations yield varying levels of photosynthetic growth (Ile, Val, Ala, Leu, Met>Thr, Cys, Ser). Six of these mutations lead to varying atrazine resistance (Met, Leu > Ser, Ala, Thr, Cys>Val, He). None of the mutations lead to atrazine sensitivity. These results suggest that hydrophobic residues of moderate size function best at this position. This set of mutations at isoleucine L229 include the first examples of mutants with null quinone function and the first examples of crystal- lography-based genetic engineering in the design of herbicide resistant reaction centers.