Phylogenetic Engineering of the Ribulose-1,5-bisphosphate Carboxylase/Oxygenase Large Subunit in <i>Chlamydomonas Reinhardtii</i>

Thirty-four residues in the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) may account for the kinetic differences between Rubisco enzyme from green algae and land plants. By substituting these "phylogenetic residues" as groups and combinations of groups in the large subunit of the green alga Chlamydomonas reinhardtii with those of land-plant Rubisco, the functions and relationships of these "phylogenetic groups" were determined. A phylogenetic-group substitution at the base of catalytic loop 6 of the large subunit decreases the CO 2 /O 2 specificity of the enzyme, but function is restored by a further phylogenetic-group substitution at the carboxy-terminal tail. Therefore, these two regions of the large subunit, which sandwich loop 6, are complementary. In addition, combining substitutions at the base of loop 6 and the large/small-subunit interface region produces a mutant enzyme that has to be complemented by the land-plant small subunit for function in Chlamydomonas. On the other hand, substitutions in -helix G of the large subunit reduce the holoenzyme level, and, because Chlamydomonas mutants with additional substitutions in α-helices 7 and 8 cannot be recovered as photosynthetic-transformants, the three α helices appear to influence holoenzyme assembly. A previous study showed that substituting five large-subunit residues and a small-subunit loop with land-plant identities produced an enzyme (termed "penta/ABSO") with land-plant catalytic properties. In the present study, through structural dissection, it is concluded that all the residues substituted in penta/ABSO are required for the shift towards land-plant catalysis. Among the residues substituted in penta/ABSO is methyl-Cys-256, which indicates that posttranslational modifications of the large subunit may also play a role in catalysis. Further study of cysteine methylation and proline hydroxylation showed that mutations of methyl-Cys-256 and hydroxy-Pro-104 influence catalysis. The current study complements previous knowledge about Rubisco, and provides further structural targets for the beneficial engineering of Rubisco. iv ACKNOWLEDGEMENTS I am grateful to my Ph. D. advisor, Dr. Robert J. Spreitzer, for providing the opportunity to undertake such an interesting and challenging project. His generosity, patience, and guidance have been essential to the success of this research, and for my development as a scientist. I am also thankful to my committee members, Drs. for their time and willingness to evaluate the research as it progressed, especially to Drs. Stone and Wilson for critically reading this dissertation. Of course, this research would not be possible without the previous work conducted by past members in our laboratory. Special thanks is due to …