A Carbon-Nitrogen Lyase from Leucaena leucocephala Catalyzes the First Step of Mimosine Degradation      

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 59-phosphate but not a-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 59-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 K m and V max values of 1.16 3 10 24 M and 5.05 3 10 25 mol s 21 mg 21 , 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.

Plant Secondary Metabolites as Defenses, Regulators, and Primary Metabolites: The Blurred Functional Trichotomy

“…Detailed biochemical characterization and metabolic profiling of secondary-metabolite mutants, combined with complementation and labelling experiments, are required to further corroborate the potential roles of secondary metabolites in primary metabolism and to identify additional secondary metabolites that serve as primarymetabolite precursors. Degradation of many different secondary metabolites has been observed under specific environmental conditions (Negi et al, 2014;Zipor et al, 2015). Furthermore, alterations in primary metabolites are observed in various plants with altered secondary metabolism (Mayer et al, 2001;Narayanan et al, 2011;Huber et al, 2016;Machado et al, 2017;Zhang et al, 2020), and the accumulation of specific secondary metabolites has been associated with storage and growth in micro-evolutionary studies (Heath et al, 2014).…”

The Chemistry and Biological Activities of Mimosine: A Review

“…Mimosinase is suggested to have a homotetrameric structure (Pandey and Dwivedi, ). Because of the differences in mimosinase purified from different tissues, this enzyme can exist in more than one isoform that is expressed at different developmental stages (Negi et al ., ). Moreover, it seems that mimosinase can be encoded by a single gene.…”

“…Interestingly, mimosinase may function in channeling mimosine into other biochemical pathways including amino acid synthesis during stress conditions. Along with other downstream enzymes, mimosinase may possess a certain mechanism for utilization of mimosine as a nutrient source under drought stress conditions (Negi et al ., ). Amino acid sequence analysis indicates that mimosinase has a high similarity with cystathionine beta‐lyase (CBL) (56–70%) (Negi et al ., , ; Tawata et al ., ).…”

“…Along with other downstream enzymes, mimosinase may possess a certain mechanism for utilization of mimosine as a nutrient source under drought stress conditions (Negi et al ., ). Amino acid sequence analysis indicates that mimosinase has a high similarity with cystathionine beta‐lyase (CBL) (56–70%) (Negi et al ., , ; Tawata et al ., ). Along with the conserved active site residues of CBL, mimosinase may have a similar catalytic mode of action as CBL which involves the pathway of Met biosynthesis and cleavage of cystathionine (Breitinger et al ., ).…”

Physicochemical composition and ruminal degradability of leucaena ensiled with different levels of buriti fruit peel

“…The plant's protein‐rich foliage and tolerance to various abiotic and biotic stresses makes leucaena a highly nutritious and valuable legume for use in ruminant feed (Negi et al . ). However, these plants have low levels of soluble carbohydrates, which are essential for the development and proliferation of fermenting bacteria.…”