Abstract
The tree legume Leucaena leucocephala contains a large amount of a toxic nonprotein aromatic amino acid, mimosine, and also an enzyme, mimosinase, for mimosine degradation. In this study, we isolated a 1,520-bp complementary DNA (cDNA) for mimosinase from L. leucocephala and characterized the encoded enzyme for mimosine-degrading activity. The deduced amino acid sequence of the coding region of the cDNA was predicted to have a chloroplast transit peptide. The nucleotide sequence, excluding the sequence for the chloroplast transit peptide, was codon optimized and expressed in Escherichia coli. The purified recombinant enzyme was used in mimosine degradation assays, and the chromatogram of the major product was found to be identical to that of 3-hydroxy-4-pyridone (3H4P), which was further verified by electrospray ionization-tandem mass spectrometry. The enzyme activity requires pyridoxal 5'-phosphate but not α-keto acid; therefore, the enzyme is not an aminotransferase. In addition to 3H4P, we also identified pyruvate and ammonia as other degradation products. The dependence of the enzyme on pyridoxal 5'-phosphate and the production of 3H4P with the release of ammonia indicate that it is a carbon-nitrogen lyase. It was found to be highly efficient and specific in catalyzing mimosine degradation, with apparent Km and Vmax values of 1.16×10(-4) m and 5.05×10(-5) mol s(-1) mg(-1), respectively. The presence of other aromatic amino acids, including l-tyrosine, l-phenylalanine, and l-tryptophan, in the reaction did not show any competitive inhibition. The isolation of the mimosinase cDNA and the biochemical characterization of the recombinant enzyme will be useful in developing transgenic L. leucocephala with reduced mimosine content in the future.
Highlights
The tree legume Leucaena leucocephala contains a large amount of a toxic nonprotein aromatic amino acid, mimosine, and an enzyme, mimosinase, for mimosine degradation
These complementary DNA (cDNA) clones represent either L. leucocephala-specific genes that are missing in A. confusa or genes that are highly expressed in L. leucocephala
We expected the mimosine-degrading enzyme from L. leucocephala to be a C-N lyase for two reasons: (1) Smith and Fowden (1966) showed that a C-N lyase from L. leucocephala had mimosine-degrading activity; and (2) recently, we have found that the mimosine degradation geneD of the L. leucocephala symbiont Rhizobium sp. strain TAL1145 encodes a C-N lyase that degrades mimosine into 3H4P, pyruvate, and ammonia (Negi et al, 2013)
Summary
The tree legume Leucaena leucocephala contains a large amount of a toxic nonprotein aromatic amino acid, mimosine, and an enzyme, mimosinase, for mimosine degradation. The protein-rich foliage and tolerance to various abiotic and biotic stresses make L. leucocephala a promising legume for use as a fodder In spite of these desirable attributes, the use of L. leucocephala as a fodder is rather limited because its foliage contains an N-heterocyclic nonprotein amino acid, known as mimosine, which is toxic to both prokaryotic cells (Soedarjo et al, 1994) and eukaryotic cells (Lalande, 1990). Smith and Fowden (1966) identified the mimosine-degrading enzyme from L. leucocephala seedling extracts as a carbon-nitrogen (C-N) lyase that converted mimosine into 3,4-dihydroxypyridine (3,4DHP), pyruvic acid, and ammonia (Fig. 1). The goals of this study were to isolate complementary DNA (cDNA) for a mimosine-degrading enzyme from L. leucocephala and to determine the biochemical and kinetic properties of the encoded enzyme This will help us to understand roles of mimosine and mimosine-degrading enzymes in L. leucocephala. It may be useful in developing transgenic L. leucocephala with reduced mimosine content, which will make this tree legume suitable for use as a nutritious fodder for animals in the future
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
