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Isolation and Characterization of Novel β-Cyanoalanine Synthase and Cysteine Synthase Genes from Cassava

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Abstract

Cassava is an important staple food crop, feeding 600 million people worldwide, which produce cyanogenic glycosides. Cyanogenic glycosides in cassava are known to act as a deterrent for herbivores as well as serve as a mobile source of reduced nitrogen. Cassava is also equipped with a cyanide detoxification pathway, mediated by β-cyanoalanine synthase (β-CAS) which converts cyanide into asparagine. β-CAS, belonging to the Bsas family of enzymes, is multi functional and shares sequence homology with cysteine synthase (CS). Using rapid amplification of cDNA end-polymerase chain reaction (RACE-PCR), two cDNA sequences were isolated from cassava. The two clones named MANes;BsasA (accession no. EU350583) and MANes;BsasB (accession no. HQ257219), showed high homology to known β-CAS enzymes (80% and 75% amino acid similarity to Arabidopsis and 76% and 82% similarity to spinach, respectively). The kinetic properties of the two clones were characterized in a Escherichia coli NK3 mutant strain which lacks activity for any of the Bsas proteins. Kinetic studies showed that MANes;BsasB is a β-CAS with a CAS/CS activity ratio of 72 while MANes;BsasA is a CS showing bifunctional capabilities and with a CAS/CS activity ratio of 11. The isolation of cassava β-CAS and CS genes reported here paves the way for their utilization in genetically enhancing the cyanide detoxification potential of cassava and/or increase of the essential amino acid cysteine, which has been found to be low in nutritionally compromised individuals.

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Acknowledgements

The authors would like to thank Professor Kazuki Saito (Chiba University, Chiba, Japan) for providing the NK3 strain and the A. thaliana β-CAS full-length clone used in this study. Special thanks also to Dr. Timothy Porch (USDA-Tropical Agriculture Research Station, Mayaguez, PR, USA) and Dr. Argelia Lawrence (Arkansas State University, Jonesboro, AR, USA) for their critical review of this manuscript. This study was funded by grant to DS from the Research Initiation Program of the National Science Foundation (award number IOS 0641084).

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  1. Department of Biology, University of Puerto Rico Mayaguez, Mayaguez, 00680, Puerto Rico

    Josean Marrero-Degro, Joan Marcano-Velázquez & Dimuth Siritunga

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  1. Josean Marrero-Degro
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  2. Joan Marcano-Velázquez
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  3. Dimuth Siritunga
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Correspondence to Dimuth Siritunga.

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Marrero-Degro, J., Marcano-Velázquez, J. & Siritunga, D. Isolation and Characterization of Novel β-Cyanoalanine Synthase and Cysteine Synthase Genes from Cassava. Plant Mol Biol Rep 29, 514–524 (2011). https://doi.org/10.1007/s11105-010-0255-4

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