Characterization of a plant S-adenosylmethionine synthetase from Acacia koa.

The Acacia koa S-adenosylmethionine (SAM) synthetase was identified from transcriptome data and cloned into the T7-expression vector pEt14b. Assays indicate a thermoalkaliphic enzyme which tolerates conditions up to pH 10.5, 55 °C and 3 M KCl. In vitro examples of plant SAM-synthetase activity are scarce, however this study provides supporting evidence that these extremophilic properties may actually be typical for this plant enzyme. Enzyme kinetic constants (Km = 1.44 mM, Kcat = 1.29 s-1, Vmax 170 µM. min-1) are comparable to nonplant SAM-synthetases except that substrate inhibition was not apparent at 10 mM ATP/L-methionine. Methods were explored in this study to reduce feedback inhibition, which is known to limit SAM-synthetase activity in vitro. Four single-point mutation variants of the Acacia koa SAM-synthetase were produced, each with varying degrees of reduced reaction rate, greater sensitivity to product inhibition and loss of thermophilic properties. Although an enhanced mutant was not produced, this study describes the first mutagenesis of a plant SAM-synthetase. Overcoming feedback inhibition was accomplished by the addition of organic solvent to enzyme assays. Acetonitrile, methanol or dimethylformamide, when included as 25% of the assay volume, improved total SAM production by 30-65%.

Methods of Mimosine Extraction from Leucaena leucocephala (Lam.) de Wit Leaves.

Mimosine is a nonprotein amino acid biosynthesized from OAS (O-acetylserine) and 3H4P (3-hydroxy-4-pyridone or its tautoisomer 3,4-dihydroxypyridine). This amino acid constitutively occurs in all parts of Leucaena leucocephala (Lam.) de Wit plants and is found at higher concentrations in seeds and leaves. This metabolite has several useful activities, such as antioxidant, allelochemical, insecticidal, antimicrobial, metal chelating, and antitumor. Mimosine is well studied in biomedical research due its ability to inhibit cells in the late G1 phase and to induce cell apoptosis. Two simple methods of mimosine extraction from leucaena leaves, pulverized and whole maceration, are described herein in detail.

Heterologous expression and characterization of a thermoalkaliphilic SAM-synthetase from giant leucaena (Leucaena leucocephala subsp glabrata).

The cDNA encoding S-adenosylmethionine (SAM) synthetase was isolated from giant leucaena (Leucaena leucocephala subsp. glabrata) root tissue mRNA. Transcriptome data and 5'-RLM-RACE were used to obtain the transcript sequence and clone into the T7-expression vector pEt14b. N-terminal Histidine-tagged recombinant protein was expressed highly in Escherichia coli, purified and characterized by activity assays. A straightforward method using isocratic reverse-phase HPLC analysis (mobile phase: 0.02M o-phosphoric acid) of enzyme assays determined optimal enzyme activity at pH 10.0, 55 °C and 200 mM KCl. In addition to thermophilic activity, giant leucaena SAM-synthetase remains highly active in solutions containing up to 4 M KCl and accepts Na+ to some extent as a substitute for K+, a known required cofactor for SAM-synthetases. The enzyme followed Michaelis-Menten kinetics (Km = 1.82 mM, Kcat = 1.17 s-1, Vmax 243.9 µM. min-1) and was not inhibited by spermidine, spermine or nicotianamine. Giant leucaena SAM-synthetase is a highly tolerant enzyme to extreme conditions, suggesting further studies on plant SAM-synthetases.

Biochemistry of plants N-heterocyclic non-protein amino acids.

Plants catalyze the biosynthesis of a large number of non-protein amino acids, which are usually toxic for other organisms. In this review, the chemistry and metabolism of N-heterocyclic non-protein amino acids from plants are described. These N-heterocyclic non-protein amino acids are composed of ß-substituted alanines and include mimosine, ß-pyrazol-1-yl-L-alanine, willardiine, isowillardiine, and lathyrine. These ß-substituted alanines consisted of an N-heterocyclic moiety and an alanyl side chain. This review explains how these individual moieties are derived from their precursors and how they are used as the substrate for biosynthesizing the respective N-heterocyclic non-protein amino acids. In addition, known catabolism and possible role of these non-protein amino acids in the actual host is explained.

Mimosine accumulation in Leucaena leucocephala in response to stress signaling molecules and acute UV exposure.

Mimosine is a non-protein amino acid of Fabaceae, such as Leucaena spp. and Mimosa spp. Several relevant biological activities have been described for this molecule, including cell cycle blocker, anticancer, antifungal, antimicrobial, herbivore deterrent and allelopathic activities, raising increased economic interest in its production. In addition, information on mimosine dynamics in planta remains limited. In order to address this topic and propose strategies to increase mimosine production aiming at economic uses, the effects of several stress-related elicitors of secondary metabolism and UV acute exposure were examined on mimosine accumulation in growth room-cultivated seedlings of Leucaena leucocephala spp. glabrata. Mimosine concentration was not significantly affected by 10 ppm salicylic acid (SA) treatment, but increased in roots and shoots of seedlings treated with 84 ppm jasmonic acid (JA) and 10 ppm Ethephon (an ethylene-releasing compound), and in shoots treated with UV-C radiation. Quantification of mimosine amidohydrolase (mimosinase) gene expression showed that ethephon yielded variable effect over time, whereas JA and UV-C did not show significant impact. Considering the strong induction of mimosine accumulation by acute UV-C exposure, additional in situ ROS localization, as well as in vitro antioxidant assays were performed, suggesting that, akin to several secondary metabolites, mimosine may be involved in general oxidative stress modulation, acting as a hydrogen peroxide and superoxide anion quencher.

An O-acetylserine (thiol) lyase from Leucaena leucocephala is a cysteine synthase but not a mimosine synthase.

In plants, the final step of cysteine formation is catalyzed by O-acetylserine (thiol) lyase (OAS-TL). The purpose of this study was to isolate and characterize an OAS-TL from the tree legume Leucaena leucocephala (leucaena). Leucaena contains a toxic, nonprotein amino acid, mimosine, which is also formed by an OAS-TL, and characterization of this enzyme is essential for developing a mimosine-free leucaena for its use as a protein-rich fodder. The cDNA for a cytosolic leucaena OAS-TL isoform was obtained through interspecies suppression subtractive hybridization. A 40-kDa recombinant protein was purified from Escherichia coli and used in enzyme activity assays where it was found to synthesize only cysteine. The enzyme followed Michaelis-Menten kinetics, and the Km was calculated to be 1,850±414 µM sulfide and the Vmax was 200.6±19.92 µM cysteine min(-1). The N-terminal affinity His-tag was cleaved from the recombinant OAS-TL to eliminate its possible interference in binding with the substrate, 3-hydroxy-4-pyridone, for mimosine formation. The His-tag-cleaved OAS-TL was again observed to catalyze the formation of cysteine but not mimosine. Thus, the cytosolic OAS-TL from leucaena used in this study is specific for only cysteine synthesis and is different from previously reported OAS-TLs that also function as ß-substituted alanine synthases.

A carbon-nitrogen lyase from Leucaena leucocephala catalyzes the first step of mimosine degradation.

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.

Expression of bacterial genes in transgenic tobacco: methods, applications and future prospects

Tobacco is the most commonly used plant for expression of transgenes from a variety of organisms, because it is easily grown and transformed, it provides abundant amounts of fresh tissue and has a well-established cell culture system. Many bacterial proteins involved in the synthesis of commercial products are currently engineered for production in tobacco. Bacterial enzymes synthesized in tobacco can enhance protection against abiotic stresses and diseases, and provide a system to test applied strategies such as phytoremediation. Examples of bacterial gene expression in tobacco include production of antigen proteins from several human bacterial pathogens as vaccines, bacterial proteins for enhancing resistance against insects, pathogens and herbicides, and bacterial enzymes for the production of polymers, sugars, and bioethanol. Further improvements in the expression of recombinant proteins and their recovery from tobacco will enhance production and commercial use of these proteins. This review highlights the dynamic use of tobacco in bacterial protein production by examining the most relevant research in this field.

Expression of bacterial genes in transgenic tobacco: methods, applications and future prospects.

Tobacco is the most commonly used plant for expression of transgenes from a variety of organisms, because it is easily grown and transformed, it provides abundant amounts of fresh tissue and has a well-established cell culture system. Many bacterial proteins involved in the synthesis of commercial products are currently engineered for production in tobacco. Bacterial enzymes synthesized in tobacco can enhance protection against abiotic stresses and diseases, and provide a system to test applied strategies such as phytoremediation. Examples of bacterial gene expression in tobacco include production of antigen proteins from several human bacterial pathogens as vaccines, bacterial proteins for enhancing resistance against insects, pathogens and herbicides, and bacterial enzymes for the production of polymers, sugars, and bioethanol. Further improvements in the expression of recombinant proteins and their recovery from tobacco will enhance production and commercial use of these proteins. This review highlights the dynamic use of tobacco in bacterial protein production by examining the most relevant research in this field.