Post-translational modification of the large subunit (LS) and small subunit (SS) of Rubisco and the molecular utility of Rubisco LSMT to deliver carbonic anhydrase to the active site vicinity of Rubisco

Research endeavors were first directed to obtain a Rubisco alpha N-methyltransferase (SSMT) enzyme source of high purity in order to characterize the enzyme responsible for methylation of the processed form of Rubisco SS. A partially purified enzyme was obtained, and used to conduct a brief biochemical characterization of Rubisco SSMT activity. Next, in an effort to evaluate the in vivo consequences of formation of trimethyllysine-14 in the large subunit (LS) of Rubisco, a cDNA for pea Rubisco large subunit epsilonN-Methyltransferase (Rubisco LSMT) was introduced into wheat, a species that normally contains des (methyl) Rubisco, and also the modification at trimethyllysine-14 was removed in vivo from tobacco Rubisco by RNAi-mediated gene silencing. The introduction of pea Rubisco LSMT into wheat plants with des (methyl) Rubisco resulted in the stoichiometric formation of a trimethyllysyl residue at position 14 in the LS, and Rubisco LSMT was removed from tobacco plants which resulted in the absence of formation of trimethyllysine-14 in the LS. Additionally, these experiments provide facile access to a novel form of Rubisco altered by in vivo protein processing events. Finally, a human form of carbonic anhydrase (hCAII) was chemically cross-linked with pea Rubisco LSMT. The complex between hCAII and Rubisco LSMT retained the tight and specific binding affinity for Rubisco. Moreover, binding of this complex to Rubisco, and the localization of carbonic anhydrase to the active site vicinity of Rubisco, resulted in a significant increase in the carboxylation efficiency of Rubisco manifested as a decrease in the KM (CO2) from 35 muM to 7.9 muM. These results suggest that the limitations on carbon fixation in C3 plants imposed by Rubisco activity may be surmountable in vivo.