Isolation and characterization of mimosine degrading enzyme from Arthrobacter sp. Ryudai-S1.

Leucaena leucocephala growing in the tropics and subtropics serves as potential forage for livestock because its foliage is rich in protein, fiber, and minerals. However, its use for livestock feed has been hindered by toxic nonprotein amino acid mimosine. Therefore, it is necessary to develop a method to reduce or eliminate mimosine from foliage. A previous study found that the fermentation of L. leucocephala foliage reduced the mimosine content and prompted the authors to isolate potent mimosine degrading microorganisms and characterize the mimosinase for the complete elimination of mimosine in the L. leucocephala foliage. The soil screening of the L. leucocephala tree surroundings led to the isolation of Arthrobacter sp. Ryudai-S1, which can degrade and assimilate mimosine as a nitrogen and carbon source. Mimosinase in this strain was found to be thermostable and showed strong activity. Docking model's inspection and the interaction energy calculation between mimosine-pyridoxal-5'-phosphate (PLP) complex and the active site of this enzyme identified 11 important amino acid residues that stabilized the binding. Of these amino acid residues, mutation experiment suggested that Tyr-263' and Phe-34 stabilizes the substrate binding and play a critical role in guiding the substrate to proper positions to accomplish high catalytic efficacy and selectivity. These observations suggest that Arthrobacter sp. Ryudai-S1 could be potentially useful for the development of L. leucocephala feed with reduced mimosine content.

In vitro rumen degradability of tropical legumes and their secondary metabolites depends on inoculum source.

In this study, the in vitro apparent rumen degradability of organic matter (ARDOM) and plant secondary metabolites (ARDPSM) of three tropical legumes (Mucuna pruriens, Canavalia ensiformis, and Leucaena leucocephala) were assessed. For this, 3 experiments were set up, i.e., single end-point incubations (24 h) with ruminal inoculum from either Belgian or Cuban sheep, as well as kinetic assessments (0 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, and 24 h) inoculum from Belgian sheep. L-mimosine, L-canavanine, Concanavalin A (Con A), and trypsin inhibitor (TI) were the plant secondary metabolites (PSM) targeted in this study. In all three experiments, both beans, as well as forage/bean meals of M. pruriens and C. ensiformis and their PSM, were extensively degraded during 24 h incubation, irrespective of the inoculum source (0.44 to 0.70 and 0.43 to 0.78 g/g of organic matter (OM) for ARDOM, respectively, and > 0.80 g/g for L-canavanine, > 0.76 TIU/TIU for TI, and > 0.95 g/g for Con A, for both legumes). Forage meal of L. leucocephala was considerably less degraded, with apparent ruminal degradabilities of 0.20 g/g OM and 0.35 g/g OM after 24 h incubation with Belgian or Cuban sheep inoculum, respectively. This could - at least partially - be related to L-mimosine, present in L. leucocephala, which was hardly degraded in the Belgian incubation, while a more extensive ruminal breakdown was observed under the Cuban conditions (0.05 g/g PSM vs. 0.78 g/g PSM, respectively). The negative effect of L-mimosine on OM degradability was supported in an additional in vitro experiment with straw and inoculum from Belgian sheep, as ruminal degradation of straw was 31% lower when pure L-mimosine was supplemented.

Molecular characterization of cytosolic cysteine synthase in Mimosa pudica.

In the cysteine and mimosine biosynthesis process, O-acetyl-L-serine (OAS) is the common substrate. In the presence of O-acetylserine (thiol) lyase (OASTL, cysteine synthase) the reaction of OAS with sulfide produces cysteine, while with 3-hydroxy-4-pyridone (3H4P) produces mimosine. The enzyme OASTL can either catalyze Cys synthesis or both Cys and mimosine. A cDNA for cytosolic OASTL was cloned from M. pudica for the first time containing 1,410 bp nucleotides. The purified protein product from overexpressed bacterial cells produced Cys only, but not mimosine, indicating it is Cys specific. Kinetic studies revealed that pH and temperature optima for Cys production were 6.5 and 50 °C, respectively. The measured Km, Kcat, and Kcat Km-1 values were 159 ± 21 µM, 33.56 s-1, and 211.07 mM-1s-1 for OAS and 252 ± 25 µM, 32.99 s-1, and 130.91 mM-1s-1 for Na2S according to the in vitro Cys assay. The Cy-OASTL of Mimosa pudica is specific to Cys production, although it contains sensory roles in sulfur assimilation and the reduction network in the intracellular environment of M. pudica.

Isolation and characterization of mimosine, 3, 4 DHP and 2, 3 DHP degrading bacteria from a commercial rumen inoculum.

The presence of the toxic amino acid mimosine in Leucaena leucocephala restricts its use as a protein source for ruminants. Rumen bacteria degrade mimosine to 3,4- and 2,3-dihydroxypyridine (DHP), which remain toxic. Synergistes jonesii is believed to be the main bacterium responsible for degradation of these toxic compounds but other bacteria may also be involved. In this study, a commercial inoculum provided by the Queensland's Department of Agriculture, Fisheries, and Forestry was screened for isolation and characterization of mimosine, 3,4- and 2,3-DHP degrading bacterial strains. A new medium for screening of 2,3-DHP degrading bacteria was developed. Molecular and biochemical approaches used in this study revealed four bacterial isolates - Streptococcus lutetiensis, Clostridium butyricum, Lactobacillus vitulinus, and Butyrivibrio fibrisolvens - to be able to completely degrade mimosine within 7 days of incubation. It was also observed that C. butyricum and L. vitulinus were able to partially degrade 2,3-DHP within 12 days of incubation, while S. lutetiensis, was able to fully degrade both 3,4 and 2,3 DHP. Collectively, we concluded that S. jonesii is not the sole bacterium responsible for detoxification of Leucaena. Comprehensive screening of rumen fluid of cattle grazing on Leucaena pastures is needed to identify additional mimosine-detoxifying bacteria and contribute to development of more effective inoculums to be used by farmers against Leucaena toxicity.

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.

Systemic effects of Leucaena leucocephala ingestion on ringtailed lemurs (Lemur catta) at Berenty Reserve, Madagascar.

Leucaena (Leucaena leucocephala) is a leguminous tree that is nutritious forage for domestic livestock when ingested in limited amounts. Unfortunately, leucaena contains mimosine, a plant amino acid, that can be toxic when ingested at higher concentrations. Reported toxic effects include alopecia (fur loss), poor body condition, infertility, low birth weight, thyroid gland dysfunction, and organ toxicity. Originally native to Mexico and Central America, leucaena has been introduced throughout the tropics, including Berenty Reserve, Madagascar where it was planted as supplemental browse for livestock. In Berenty, a seasonal syndrome of alopecia in ringtailed lemurs (Lemur catta) is associated with eating leucaena. Although much is known about the toxic effects of leucaena and mimosine on domestic animals and humans, the systemic effects on wildlife had not been studied. In a comparison of lemurs that include leucaena in their diet and those that do not, we found that animals that ingest leucaena absorb mimosine but that ingestion does not affect body condition, cause kidney or liver toxicity, or affect the intestinal tract. Alopecia is due to mimosine's interference of the hair follicle cycle. Leucaena ingestion is associated with higher serum albumin, α-tocopherol, and thyroxine concentrations, suggesting that leucaena may provide some nutritional benefit and that lemurs can detoxify and convert mimosine to a thyroid stimulating metabolite. The primary conservation consequence of leucaena ingestion at Berenty may be increased infant mortality due to the infants' inability cling to their alopecic mothers. The widespread introduction of leucaena throughout the tropics and its rapid spread in secondary forest conditions mean that many other leaf-eating mammals may be including this tree in their diet. Thus, exposure to leucaena should be considered when wildlife health is being evaluated, and the potential effects on wildlife health should be considered when contemplating leucaena introduction into or near wildlife habitat.

midD-encoded 'rhizomimosinase' from Rhizobium sp. strain TAL1145 is a C-N lyase that catabolizes L-mimosine into 3-hydroxy-4-pyridone, pyruvate and ammonia.

Rhizobium sp. strain TAL1145 catabolizes mimosine, which is a toxic non-protein amino acid present in Leucaena leucocephala (leucaena). The objective of this investigation was to study the biochemical and catalytic properties of the enzyme encoded by midD, one of the TAL1145 genes involved in mimosine degradation. The midD-encoded enzyme, MidD, was expressed in Escherichia coli, purified and used for biochemical and catalytic studies using mimosine as the substrate. The reaction products in the enzyme assay were analyzed by HPLC and mass spectrometry. MidD has a molecular mass of ~45 kDa and its catalytic activity was found to be optimal at 37 °C and pH 8.5. The major product formed in the reaction had the same retention time as that of synthetic 3-hydroxy-4-pyridone (3H4P). It was confirmed to be 3H4P by MS/MS analysis of the HPLC-purified product. The K m, V max and K cat of MidD were 1.27 × 10(-4) mol, 4.96 × 10(-5) mol s(-1) mg(-1), and 2,256.05 s(-1), respectively. Although MidD has sequence similarities with aminotransferases, it is not an aminotransferase because it does not require a keto acid as the co-substrate in the degradation reaction. It is a pyridoxal-5'-phosphate (PLP)-dependent enzyme and the addition of 50 µM hydroxylamine completely inhibited the reaction. However, the supplementation of the reaction with 0.1 µM PLP restored the catalytic activity of MidD in the reaction containing 50 µM hydroxylamine. The catalytic activity of MidD was found to be specific to mimosine, and the presence of its structural analogs including L-tyrosine, L-tryptophan and L-phenylalanine did not show any competitive inhibition. In addition to 3H4P, we also identified pyruvate and ammonia as other degradation products in equimolar quantities of the substrate used. The degradation of mimosine into a ring compound, 3H4P with the release of ammonia indicates that MidD of Rhizobium sp. strain TAL1145 is a C-N lyase.

Preparative scale isolation, purification and derivatization of mimosine, a non-proteinogenic amino acid.

Focusing on drug discovery non-proteinogenic amino acids have often been used as important building blocks for construction of compound libraries in the filed of combinatorial chemistry and chemical biology. Highly homogeneous L: -mimosine, α-amino-ß-(3-hydoxy-4-oxo-1,4-dihydropyridin-1-yl)-propanoic acid, a non-proteinogenic amino acid, has been successfully isolated and purified on an industrial scale from wild leaves of Leucaena (Leucaena leucocephala de Wit) which is a widely distributed legume in Okinawa, a sub-tropical island in Japan. Optical purity determinations used for quality control have been established through diastereomer formation. Physico-chemical properties and biological properties of purified mimosine have been clarified. Mimosine is sparingly soluble in water and organic solvents but can be dissolved in aqueous alkaline solution. The tyrosinase pathway is of particular interest in the cosmetic field, since mimosine is an analog of tyrosine. Thus the present purified mimosine have been tested in tyrosinase inhibitory assays. The IC50 for tyrosinase inhibitory activity of purified Mim was compared with kojic acid. Mimosine shows significant inhibition of melanin production in murine melanoma cells. The derivatization of mimosine has been investigated with a focus on its use in conventional peptide syntheses to generate mimosyl peptides. N-(9-Fluorenylmethoxycarbonyloxy)-mimosine and resin-bound mimosine for solid-phase syntheses have also been performed. Highly homogeneous Mim is a useful material for the development of functional cosmetics or active pharmaceutical ingredients.

Transient hypoxia-inducible factor activation in rat renal ablation and reduced fibrosis with L-mimosine.

AIM: Hypoxia-inducible factor (HIF) activity during the course of chronic kidney disease (CKD) development is poorly defined, and the effect of HIF activation on CKD is still controversial. The purpose of the present study was to characterize HIF expression during the course of CKD development, and to investigate the effect of HIF activation on CKD by using prolyl hydroxylase (PHD) inhibitor L-mimosine. METHODS: Rats with remnant kidneys (RK) were killed at week 1, 2, 4, 6, 8, 12 after subtotal nephrectomy. An additional group of RK rats was treated with L-mimosine to study the effect of HIF-α activation. RESULTS: Tubulointerstitial hypoxia in the remnant kidney began at week 1 and continued, albeit attenuated, until week 12, the last time point examined. The nuclear expression of HIF-1α and HIF-2α, as well as typical HIF target genes VEGF (vascular endothelial growth factor), HO-1 (heme oxygenase-1), GLUT-1 (glucose transporter-1) and EPO (erythropoietin), were all upregulated in the early stage of RK when renal function was stable, and returned to the basal level later, accompanied by impaired renal function and interstitial fibrosis. L-mimosine administered from week 5 to week 12 led to accumulation of HIF-1α and HIF-2α proteins, increased expression of VEGF, HO-1 and GLUT-1, and improved renal function. Furthermore, fibrosis markers α-smooth muscle actin (α-SMA) and Collagen III, as well as peritubular capillary rarefaction index, were all significantly decreased after L-mimosine treatment. CONCLUSION: There was a transient HIF-α activation in the remnant kidney of rats at the early stage following subtotal nephrectomy. L-mimosine administered in later stages re-activated HIF-α and reduced tubulointerstitial fibrosis.

Productive performance and urinary excretion of mimosine metabolites by hair sheep grazing in a silvopastoral system with high densities of Leucaena leucocephala.

The aim of this study was to evaluate daily weight gain (DWG), total dry matter (DM) intake, rumen degradability of forage, and urinary excretion of mimosine metabolites by hair sheep in a silvopastoral system with high densities of Leucaena leucocephala. A completely randomized design was carried out with two treatments: treatment 1 (T1) silvopastoral system with leucaena at a density of 35,000 plants/ha and treatment 2 (T2), leucaena at a density of 55,000 plants/ha. Leucaena was associated with tropical grasses Panicum maximum and Cynodon nlemfluensis. Twenty-four male Pelibuey lambs of 23.2 ± 3.4 kg live weight (LW) were used (12 lambs per treatment). Results showed differences (P < 0.05) in DWG of T1 (106.41 ± 11.66 g(-1) sheep(-1)) with respect to that of T2 (81.33 ± 11.81 g(-1) sheep). Voluntary intake was higher in lambs from T1 (83.81 ± 04.07 g DM/kg LW(0.75)) with respect to that from T2 (71.67 ± 8.12 g DM/kg LW(0.75)). There was a difference in color of urine between sheep of T1 and T2, the latter giving positive results for the presence of metabolites derived from mimosine (3-4 dihydroxypyridine and 2-3 dihydroxy pyridone). Rumen degradability of DM of L. leucocephala was higher (P < 0.05) compared to that of P. maximum and C. nlemfluensis (72.94 ± 0.40 vs. 67.06 ± 1.50 and 63.25 ± 1.51 %, respectively). It is concluded that grazing at high densities of L. leucocephala affects daily weight gain of hair sheep, possibly due to ingestion of high amounts of mimosine which may exert an adverse effect on voluntary intake.

A comparative study on mimosine, 3,4-dihydroxy pyridone (3,4-DHP) and 2,3-dihydroxy pyridone (2,3-DHP), purine derivatives (PD) excretion in the urine, thyroid hormone and blood metabolites profiles of Thai swamp buffalo (Bubalus bubalis) and Murrah buffalo (Bubalus bubalis).

Four Thai swamp buffaloes (SB) and four Murrah buffaloes (MB) fed a based diet of fresh ruzi grass (Bachiaria ruziziensis) with an increased proportion of fresh leucaena leaves. Intake of nutrients in animals increased when ruzi grass was mixed with leucaena. Digestibility of nutrients were the highest in SB and MB fed diets containing 25% and 50% of leucaena, respectively, and nitrogen (N) balances in both animal breeds were varied among diets. The regression equation coefficient of mimosine + DHP in the urine was twice as high in SB than in MB. Urinary purine derivatives excretion rate in SB was higher than that in MB. Plasma triiodothyronine and thyroxine declined in both animal breeds fed a diet containing >1 g mimosine intake/kg BW(0.75)/day. Plasma urea-N was the lowest in SB, but the highest in MB when fed a diet containing 84% of leucaena. Plasma ß-HBA in SB have declined when diets contained >50% of leucaena but that in MB was not affected by any diet. In conclusion, the effect of leucaena in diet upon buffalo breeds depends on the proportion of leucaena in the diet, mimosine contents and condensed tannins components.

Influence of the Nonprotein Amino Acid Mimosine in Peptide Conformational Propensities from Novel Amber Force Field Parameters.

Mimosine is a nonprotein amino acid derived from plants known for its ability to bind to divalent and trivalent metal cations such as Zn2+, Ni2+, Fe2+, or Al3+. This results in interesting antimicrobial and anticancer properties, which make mimosine a promising candidate for therapeutic applications. One possibility is to incorporate mimosine into synthetic short peptide drugs. However, how this amino acid affects the peptide structure is not well understood, reducing our ability to design effective therapeutic compounds. In this work, we used computer simulations to understand this question. We first built parameters for the mimosine residue to be used in combination with two classical force fields of the Amber family. Then, we used atomistic molecular dynamics simulations with the resulting parameter sets to evaluate the influence of mimosine in the structural propensities for this amino acid. We compared the results of these simulations with homologous peptides, where mimosine is replaced by either phenylalanine or tyrosine. We found that the strong dipole in mimosine induces a preference for conformations where the amino acid rings are stacked over more extended conformations. We validated our results using quantum mechanical calculations, which provide a robust foundation for the outcome of our classical simulations.

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.

Characterization of a thermophilic cytochrome P450 of the CYP203A subfamily from Binh Chau hot spring in Vietnam.

Cytochromes P450 (CYPs or P450s) comprise a superfamily of heme-containing monooxygenases that are involved in a variety of biological processes. CYPs have broad utilities in industry, but most exhibit low thermostability, limiting their use on an industrial scale. Highly thermostable enzymes can be obtained from thermophiles in geothermal areas, including hot springs, offshore oil-producing wells and volcanoes. Here, we report the identification of a gene encoding for a thermophilic CYP from the Binh Chau hot spring metagenomic database, which was designated as P450-T2. The deduced amino acid sequence showed the highest identity of 73.15% with CYP203A1 of Rhodopseudomonas palustris, supporting that P450-T2 is a member of the CYP203A subfamily. Recombinant protein expression yielded 541 nm. The optimal temperature and pH of P450-T2 were 50 °C and 8.0, respectively. The half-life of P450-T2 was 50.2 min at 50 °C, and its melting temperature was 56.80 ± 0.08 °C. It was found to accept electrons from all tested redox partners systems, with BmCPR-Fdx2 being the most effective partner. Screening for putative substrates revealed binding of phenolic compounds, such as l-mimosine and emodin, suggesting a potential application of this new thermophilic P450 in the production of the corresponding hydroxylated products.

Oestrogenic activity of mimosine on MCF-7 breast cancer cell line through the ERα-mediated pathway.

Hormone replacement therapy has been a conventional treatment for postmenopausal symptoms in women. However, it has potential risks of breast and endometrial cancers. The aim of this study was to evaluate the oestrogenicity of a plant-based compound, mimosine, in MCF-7 cells by in silico model. Cell viability and proliferation, ERα-SRC1 coactivator activity and expression of specific ERα-dependent marker TFF1 and PGR genes were evaluated. Binding modes of 17ß-oestradiol and mimosine at the ERα ligand binding domain were compared using docking and molecular dynamics simulation experiments followed by binding interaction free energy calculation with molecular mechanics/Poisson-Boltzmann surface area. Mimosine showed increased cellular viability (64,450 cells/ml) at 0.1 µM with significant cell proliferation (120.5%) compared to 17ß-oestradiol (135.2%). ER antagonist tamoxifen significantly reduced proliferative activity mediated by mimosine (49.9%). Mimosine at 1 µM showed the highest ERα binding activity through increased SRC1 recruitment at 186.9%. It expressed TFF1 (11.1-fold at 0.1 µM) and PGR (13.9-fold at 0.01 µM) genes. ERα-mimosine binding energy was -49.9 kJ/mol, and it interacted with Thr347, Gly521 and His524 of ERα-LBD. The results suggested that mimosine has oestrogenic activity.

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.

Pyruvate carboxylase is involved in metabolism of mimosine by Rhizobium sp. strain TAL1145.

The objective of this study was to determine the role of midK, which encodes a protein similar to pyruvate carboxylase, in mimosine degradation by Rhizobium sp. strain TAL1145. The midK gene is located downstream of midR in the cluster of genes for mimosine degradation in Rhizobium sp. strain TAL1145. The midK mutants of TAL1145 degraded mimosine slower than the wild-type. These mutants could utilize pyruvate as a source of carbon, indicating that there is another pyruvate carboxylase (pyc) gene in TAL1145. Two classes of clones were isolated from the library of TAL1145 by complementing a pyc mutant of Rhizobium etli, one class contained midK, while the other carried pyc. Both midK and pyc of TAL1145 complemented the midK mutant for mimosine degradation, and also the R. etli pyc mutant for pyruvate utilization. The midK-encoded pyruvate carboxylase was required for an efficient conversion of mimosine into 3-hydroxy-4-pyridone (HP).

G1/S cell cycle arrest provides anoikis resistance through Erk-mediated Bim suppression.

Proper attachment to the extracellular matrix is essential for cell survival. Detachment from the extracellular matrix results in an apoptotic process termed anoikis. Anoikis induction in MCF-10A mammary epithelial cells is due not only to loss of survival signals following integrin disengagement, but also to consequent downregulation of epidermal growth factor (EGFR) and loss of EGFR-induced survival signals. Here we demonstrate that G(1)/S arrest by overexpression of the cyclin-dependent kinase inhibitors p16(INK4a), p21(Cip1), or p27(Kip1) or by treatment with mimosine or aphidicolin confers anoikis resistance in MCF-10A cells. G(1)/S arrest-mediated anoikis resistance involves suppression of the BH3-only protein Bim. Furthermore, in G(1)/S-arrested cells, Erk phosphorylation is maintained in suspension and is necessary for Bim suppression. Following G(1)/S arrest, known proteins upstream of Erk, including Raf and Mek, are not activated. However, retained Erk activation under conditions in which Raf and Mek activation is lost is observed, suggesting that G(1)/S arrest acts at the level of Erk dephosphorylation. Thus, anoikis resistance by G(1)/S arrest is mediated by a mechanism involving Bim suppression through maintenance of Erk activation. These results provide a novel link between cell cycle arrest and survival, and this mechanism could contribute to the survival of nonreplicating, dormant tumor cells that avert apoptosis during early stages of metastasis.

The cloned 1-aminocyclopropane-1-carboxylate (ACC) deaminase gene from Sinorhizobium sp. strain BL3 in Rhizobium sp. strain TAL1145 promotes nodulation and growth of Leucaena leucocephala.

The objective of this study was to determine the role of 1-aminocyclopropane-1-carboxylate (ACC) deaminase of symbionts in nodulation and growth of Leucaena leucocephala. The acdS genes encoding ACC deaminase were cloned from Rhizobium sp. strain TAL1145 and Sinorhizobium sp. BL3 in multicopy plasmids, and transferred to TAL1145. The BL3-acdS gene greatly enhanced ACC deaminase activity in TAL1145 compared to the native acdS gene. The transconjugants of TAL1145 containing the native or BL3 acdS gene could grow in minimal media containing 1.5mM ACC, whereas BL3 could tolerate up to 3mM ACC. The TAL1145 acdS gene was inducible by mimosine and not by ACC, while the BL3 acdS gene was highly inducible by ACC and not by mimosine. The transconjugants of TAL1145 containing the native- and BL3-acdS genes formed nodules with greater number and sizes, and produced higher root mass on L. leucocephala than by TAL1145. This study shows that the introduction of multiple copies of the acdS gene increased ACC deaminase activities of TAL1145 and enhanced its symbiotic efficiency on L. leucocephala.

Cytosolic Cysteine Synthase Switch Cysteine and Mimosine Production in Leucaena leucocephala.

In higher plants, multiple copies of the cysteine synthase gene are present for cysteine biosynthesis. Some of these genes also have the potential to produce various kinds of ß-substitute alanine. In the present study, we cloned a 1275-bp cDNA for cytosolic O-acetylserine(thiol)lyase (cysteine synthase) (Cy-OASTL) from Leucaena leucocephala. The purified protein product showed a dual function of cysteine and mimosine synthesis. Kinetics studies showed pH optima of 7.5 and 8.0, while temperature optima of 40 and 35 °C, respectively, for cysteine and mimosine synthesis. The kinetic parameters such as apparent Km, kcat were determined for both cysteine and mimosine synthesis with substrates O-acetylserine (OAS) and Na2S or 3-hydroxy-4-pyridone (3H4P). From the in vitro results with the common substrate OAS, the apparent kcat for Cys production is over sixfold higher than mimosine synthesis and the apparent Km is 3.7 times lower, suggesting Cys synthesis is the favored pathway.