The tree-legume Leucaena leucocephala (leucaena) contains a large amount of a toxic non-protein aromatic amino acid, mimosine, and also an enzyme, mimosinase, for mimosine degradation. In this study, we isolated a 1520-bp cDNA for mimosinase from leucaena 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 E. coli. The purified recombinant enzyme was used in mimosine degradation assays, and the chromatogram of the major product was found identical to that of... More
The tree-legume Leucaena leucocephala (leucaena) contains a large amount of a toxic non-protein aromatic amino acid, mimosine, and also an enzyme, mimosinase, for mimosine degradation. In this study, we isolated a 1520-bp cDNA for mimosinase from leucaena 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 E. coli. The purified recombinant enzyme was used in mimosine degradation assays, and the chromatogram of the major product was found identical to that of 3-hydroxy-4-pyridone (3H4P), which was further verified by ESI-MS/MS. The enzyme activity requires pyridoxal 5'-phosphate (PLP) but not α-keto acid and 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 PLP, and production of 3H4P with the release of ammonia indicate that it is a C-N lyase. It was found to be highly efficient and specific in catalyzing mimosine degradation with the apparent Km and Vmax values of 1.16x10-4 M and 5.05x10-5 mole•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 leucaena with reduced mimosine content in the future.
