An Analysis of Enzymatic Activity in Truncated and Wild-Type Auxin Conjugate Hydrolases in Medicago Trunculata (barrel clover) and Implications in Structural Modeling
Date of Award
Master of Science (MS)
College of Science and Mathematics
Thesis Sponsor/Dissertation Chair/Project Chair
James J. Campanella
Auxins are plant phytohormones that play an important part in plant growth and development. The level of active auxins in plants is regulated by enzymes known as hydrolases that release inactive bound forms of auxins, known as conjugates, as the active free form. We have previously isolated and enzymatically characterized the auxin conjugate hydrolase family from the model legume Medicago truncatula (barrel clover). In the process of cloning the cDNA for the genes MtIAR31, -32, -33, -34, and -36, we also cloned a truncated version of MtIAR33 (AMtIAR33) that apparently expresses an alternative hydrolase protein from a secondary, internal AUG start site. We expressed this truncated protein in vitro, compared its enzymatic activity to the full-length protein, and found the activity and substrate recognition of the two differed substantially. The full-length MtIAR33 had high activity against IAA-Aspartate (-1160 pmol auxin released/min/ml) with low activity against IB A-Alanine, while AMtIAR33 lost almost all activity against IAA-Aspartate, but gained activity against IB A-Alanine (-140 pmol auxin released/min/ml). These major differences in activity suggested a possible biological importance, so we designed expression constructs containing versions of MtIAR31, -32, -34, and -36 that were each truncated at the same start site found in AMtIAR33. Our goal has been to first, analyze each of these truncated hydrolases for substrate activity/specificity, and second, to model the three-dimensional structure of the wild-type and truncated hydrolases along with substrates to better understand which structural peptide motifs are influencing the activity of these enzymes. Our results suggest the possibility that the truncated versions encode for alternatively spliced enzymes.
Sigethy, Scott, "An Analysis of Enzymatic Activity in Truncated and Wild-Type Auxin Conjugate Hydrolases in Medicago Trunculata (barrel clover) and Implications in Structural Modeling" (2007). Theses, Dissertations and Culminating Projects. 1266.