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Structure Characterization of a Peroxidase from the Windmill Palm Tree Trachycarpus fortunei

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Item Summary

Title:Structure Characterization of a Peroxidase from the Windmill Palm Tree Trachycarpus fortunei
Authors:Baker, Margaret
Keywords:palm tree
mass spectrometry
Date Issued:May 2015
Publisher:[Honolulu] : [University of Hawaii at Manoa], [May 2015]
Abstract:Plant secretory peroxidases are important commercial enzymes and play a central role in plant stress responses. The focus of this work is a unique peroxidase from the leaves of a cold tolerant palm, Trachycarpus fortunei, (i.e., windmill palm tree). Like other palm tree peroxidases, windmill palm tree peroxidase is stable at high temperatures and in the presence of denaturants. It is distinct from other palm tree peroxidases in its substrate specificity. The amino acid sequence and glycosylation had not been determined. Because glycosylation is known to play a critical role in plant peroxidase stability and activity, this knowledge is essential for structure activity studies, selection of an expression system for enzyme production, and engineering the enzyme. Glycosylation is a complex modification that is difficult to study; furthermore, there is a lack of analytical tools for characterizing plant glycoproteins. The complete amino acid sequence was determined using cDNA sequencing and biological mass spectrometry. The mature amino acid sequence is 306 residues in length. The presence of a C-terminal signal peptide predicts vacuolar targeting of the enzyme. Native windmill palm tree peroxidase was analyzed at the glycopeptide level to give a qualitative and quantitative assessment of glycosylation at each site. Windmill palm tree peroxidase has 13 sites for N-linked glycosylation, 2 of which are unique. Each site is at least partially occupied by a glycan. Major glycans are paucimannosidic, which supports the assignment of windmill palm tree peroxidase as a vacuolar peroxidase. To carry-out this work, a workflow for analyzing the glycopeptide mass spectrometry data was developed. Included in the workflow are novel tools for glycan database construction, pGlycoFilter, and validation of glycopeptide assignment, gPSMvalidator. New analytical methods are needed for the emerging field of plant glycoproteomics. The novel methods developed in this dissertation will be useful for the study of other important plant glycoproteins. This knowledge can be used to study the roles of glycosylation in this exceptionally stable and unique palm peroxidase.
Description:Ph.D. University of Hawaii at Manoa 2015.
Includes bibliographical references.
Appears in Collections: Ph.D. - Molecular Biosciences and Bioengineering

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