TitleAnalysis of the enzymological properties of prolyl-tRNA synthetases in plants focusing on the misactivation of the proline analog azetidine-2-carboxylic acid
NameLee, Jiyeon (author), Leustek, Thomas (chair), Lawton, Michael (internal member), Zilinskas, Barbara (internal member), Kerstetter, Randall (internal member), Kahn, Peter (outside member), Rutgers University, Graduate School - New Brunswick,
DescriptionAzetidine-2-carboxylic acid (A2C), a structural analogue of L-proline (Pro), inhibits the growth of bacterial, plant and animal cells. Toxicity is thought to occur when A2C is incorporated into proteins in place of Pro resulting from misrecognition by prolyl-tRNA synthetases (ProRS). Arabidopsis thaliana seedlings are highly sensitive to A2C resulting in growth inhibition. To explain this result, the activity of the two forms of ProRS from A. thaliana was studied. One form of ProRS is localized to chloroplasts/mitochondria and the other form to the cytosol. Both forms were expressed as His-tagged recombinant proteins in Escherichia coli. Purified enzymes were functionally active in the ATP-PPi exchange, and aminoacylation assays demonstrated similar Km values for Pro. A major difference was observed in the specificity for A2C. The organellar form showed a 340-fold greater Km[A2C] than the cytosolic enzyme. These results suggest that A2C-sensitivity of A. thaliana is primarily due to the inability of cytosolic ProRS to distinguish between Pro and A2C. A similar result was obtained with the ProRSs from Zea mays suggesting that the difference in substrate specificity is a conserved feature of plant cytosolic and organellar ProRSs. The tRNA-specificity of the organellar and cytosolic enzymes was also examined. The organellar ProRS At5g52520 was able to complement a conditional ProRS mutant strain of E. coli but the cytosolic ProRS At3g62120 did not. At5g52520 was able to efficiently aminoacylate both E. coli and Saccharomyces cerevisiae tRNA. In contrast, At3g62120 was able to aminoacylate S. cerevisiae tRNA more efficiently than E. coli tRNA. The interest in ProRS arose initially from the observation that some plant species, including Convallaria majalis and Polygonatum multiflorum, two members of the Liliopsida, accumulate high levels of A2C. In 1972, Norris et el. reported that ProRS from C. majalis can discriminate between Pro and A2C (Norris and Fowden, 1972). To gain a better understanding of the mechanism of A2C resistance, the cDNAs encoding ProRS were isolated from C. majalis and Polygonatum pubescens. Based on sequence homology, the cDNA encoding organellar ProRS was successfully cloned. However, attempts to produce a functional ProRS from the cloned cDNAs were unsuccessful.
NoteIncludes bibliographical references (p. 178-184)
Noteby Jiyeon Lee
CollectionGraduate School - New Brunswick Electronic Theses and Dissertations
Organization NameRutgers, The State University of New Jersey
RightsThe author owns the copyright to this work.