Characterization and Genetic Manipulation of D-cysteine Desulfhydrase from Solanum lycopersicum
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Date
2008-05-15T13:10:10Z
Authors
Todorovic, Biljana
Journal Title
Journal ISSN
Volume Title
Publisher
University of Waterloo
Abstract
Progress in DNA sequencing of plant genomes has revealed that, in addition to microorganisms, a number of plants contain genes which share similarity to microbial 1-aminocyclopropane-1-carboxylate (ACC) deaminases. ACC deaminases break down ACC, the immediate precursor of ethylene in plants, into ammonia and α-ketobutyrate. We therefore sought to isolate putative ACC deaminase cDNAs from tomato plants with the objective of establishing whether the product of this gene is a functional ACC deaminase. It was demonstrated that the enzyme encoded by the putative ACC deaminase cDNA does not have the ability to break the cyclopropane ring of ACC, but rather that it utilizes D-cysteine as a substrate, and in fact encodes a D-cysteine desulfhydrase. Kinetic characterization of the enzyme has shown that it is similar to other previously characterized D-cysteine desulfhydrases. Using site-directed mutagenesis, it was shown that altering two amino acid residues within the predicted active site changed the enzyme from D-cysteine desulfhydrase to ACC deaminase. Concomitantly, it was shown that by altering two amino acids residues at the same position within the active site of ACC deaminase from Pseudomonas putida UW4 changed this enzyme into D-cysteine desulfhydrase.
Description
Keywords
ACC deaminase, ethylene, PLP dependent enzymes, Site-directed mutagenesis