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The interconversion of ACC deaminase and d-cysteine desulfhydrase by directed mutagenesis

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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. These enzymes cleave 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. In the work reported here, 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 it utilizes d-cysteine as a substrate, and in fact encodes a d-cysteine desulfhydrase. Kinetic characterization of the tomato enzyme indicates that it is similar to other, previously characterized, d-cysteine desulfhydrases. Using site-directed mutagenesis, it was shown that altering only two amino acid residues within the predicted active site served to change the enzyme from d-cysteine desulfhydrase to ACC deaminase. Conversely, by altering two amino acid residues at the same positions within the active site of ACC deaminase from Pseudomonas putida UW4 the enzyme was converted into d-cysteine desulfhydrase. Therefore, it is possible that a change in these two residues may have occurred in an ancestral protein to result in two different enzymatic activities.

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Abbreviations

ACC:

1-Aminocyclopropane-1-carboxylate

PLP:

Pyridoxal phosphate

TRPSβ:

Tryptophan synthase β-subunit

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Acknowledgments

The work reported here was founded by a Discovery Grant from the Natural Science and Engineering Research Council of Canada to Bernard R. Glick. We thank Dr Ed Bayer, Weizmann Institute of Science, for critically reading the manuscript and Mr Zhenyu Cheng for help with mass spectrometry.

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Authors and Affiliations

  1. Department of Biology, University of Waterloo, 200 University Avenue West, Waterloo, ON, N2L 3G1, Canada

    Biljana Todorovic & Bernard R. Glick

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  1. Biljana Todorovic
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  2. Bernard R. Glick
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Correspondence to Biljana Todorovic.

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Accession numbers: The nucleotide sequence for tomato d-cysteine desulfhydrase has been deposited to GenBank Data Libraries with the accession number EU639448.

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Todorovic, B., Glick, B.R. The interconversion of ACC deaminase and d-cysteine desulfhydrase by directed mutagenesis. Planta 229, 193–205 (2008). https://doi.org/10.1007/s00425-008-0820-3

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