Substrate specificity of plant nitrilase complexes is affected by their helical twist.

Nitrilases are oligomeric, helix-forming enzymes from plants, fungi and bacteria that are involved in the metabolism of various natural and artificial nitriles. These biotechnologically important enzymes are often specific for certain substrates, but directed attempts at modifying their substrate specificities by exchanging binding pocket residues have been largely unsuccessful. Thus, the basis for their selectivity is still unknown. Here we show, based on work with two highly similar nitrilases from the plant Capsella rubella, that modifying nitrilase helical twist, either by exchanging an interface residue or by imposing a different twist, without altering any binding pocket residues, changes substrate preference. We reveal that helical twist and substrate size correlate and when binding pocket residues are exchanged between two nitrilases that show the same twist but different specificities, their specificities change. Based on these findings we propose that helical twist influences the overall size of the binding pocket.

[Improvement of Stress Resistance and Quality of Life of Adults with Nervous Restlessness after Treatment with a Passion Flower Dry Extract].

BACKGROUND: The passion flower dried ethanolic extract investigated in this non-interventional study has well-documented calmative effects and good tolerability. We investigated the effects of this extract on the stress resistance (resilience) and quality of life (QoL) of patients suffering from nervous restlessness. The addiction potential of the drug and the course of symptoms were also evaluated. METHODS: Adult patients aged ≤ 95 years with the diagnosis 'nervous restlessness' were treated for 12 weeks with a dried ethanolic extract of passion flower (Passiflora incarnata L.). Standardized questionnaires were used to evaluate the resilience (RS-13), QoL (EQ-5D including EQ-VAS), and the addiction potential (BDEPQ). RESULTS: After 12 weeks of treatment, significant (p < 0.001) improvements were measured in the patients' resilience (RS-13: from 52.1 to 67.7 points) and QoL (EQ-VAS: from 47.9 to 75.0 points). Also, the mean BDEPQ score was significantly (p < 0.001) reduced (from 23.0 to 19.3 points). The mean values of all accompanying symptoms (inner restlessness, sleep disturbance, exhaustion, fear, lack of concentration, transpiration, nausea, trembling, and palpitation) improved significantly (p < 0.001). Tolerability of treatment was rated as 'very good' or 'good' by the majority of the patients. Three cases of mild adverse events (tiredness) were reported. CONCLUSION: The passion flower extract investigated in the present study appears to be effective in improving resilience and QoL in patients suffering from nervous restlessness and is well tolerated.

Arabidopsis NITRILASE 1 Contributes to the Regulation of Root Growth and Development through Modulation of Auxin Biosynthesis in Seedlings.

Nitrilases consist of a group of enzymes that catalyze the hydrolysis of organic cyanides. They are found ubiquitously distributed in the plant kingdom. Plant nitrilases are mainly involved in the detoxification of ß-cyanoalanine, a side-product of ethylene biosynthesis. In the model plant Arabidopsis thaliana a second group of Brassicaceae-specific nitrilases (NIT1-3) has been found. This so-called NIT1-subfamily has been associated with the conversion of indole-3-acetonitrile (IAN) into the major plant growth hormone, indole-3-acetic acid (IAA). However, apart of reported functions in defense responses to pathogens and in responses to sulfur depletion, conclusive insight into the general physiological function of the NIT-subfamily nitrilases remains elusive. In this report, we test both the contribution of the indole-3-acetaldoxime (IAOx) pathway to general auxin biosynthesis and the influence of altered nitrilase expression on plant development. Apart of a comprehensive transcriptomics approach to explore the role of the IAOx route in auxin formation, we took a genetic approach to disclose the function of NITRILASE 1 (NIT1) of A. thaliana. We show that NIT1 over-expression (NIT1ox) results in seedlings with shorter primary roots, and an increased number of lateral roots. In addition, NIT1ox plants exhibit drastic changes of both free IAA and IAN levels, which are suggested to be the reason for the observed phenotype. On the other hand, NIT2RNAi knockdown lines, capable of suppressing the expression of all members of the NIT1-subfamily, were generated and characterized to substantiate the above-mentioned findings. Our results demonstrate for the first time that Arabidopsis NIT1 has profound effects on root morphogenesis in early seedling development.

Homeopathic complex remedy in the treatment of allergic rhinitis: results of a prospective, multicenter observational study.

BACKGROUND: Seasonal allergic rhinitis (SAR), also known as hay fever, is a widespread chronic respiratory disease. In treatment of SAR the use of complementary therapies is increasing, but little has been published about homeopathic complex remedies so far. Therefore, we think it is time to conduct and publish an appropriate observational study. METHODS: Course of single symptoms, impairment of quality of life, general efficacy, and tolerability of a homeopathic complex remedy containing active substances on a low dilution level have been assessed and analyzed. Altogether, 123 patients with a history of allergic rhinitis of up to 45 years have been observed for about 4 weeks. RESULTS: The majority of symptoms were shown to improve substantially and the patients' quality of life increased clearly. The overall symptom score decreased significantly from 10.3 ± 4.7 to 3.9 ± 3.1 points (p < 0.0001), and reduction of impairment of quality of life from 5.7 ± 2.3 to 1.9 ± 1.8 score points was also significant (p < 0.0001). Rating of efficacy of study medication was markedly better than efficacy rating of previous therapies (p = 0.0193). Apart from one temporary allergic reaction, the treatment was well tolerated. CONCLUSION: The homeopathic complex remedy (Pascallerg®) tested in this observational study offers a useful option in treatment of SAR in children and adults.

Herbal triplet in treatment of nervous agitation in children.

Emotional and behavioral problems in children and adolescents are no exception. To what extent a fixed plant extract combination is able to support children suffering from nervous agitation due to agitated depression among others for approximately 2 years has been investigated in a multicenter, prospective observational study (2008) with 115 children between 6 and 12 years. Assessments of the parents showed a distinct improvement in children who had attention problems, showed social withdrawal, and/or were anxious/depressive. Based on the physicians' assessment, 81.6-93.9% of the affected children had no or just mild symptoms at the end of observation concerning nine of thirteen evaluated symptoms such as depression, school/examination anxieties, further anxieties, sleeping problems, and different physical problems. Therapeutic success was not influenced by additional medication or therapies. The treatment was well tolerated. The used plant extracts have been gained from St. John's Wort herb, valerian root, and passionflower herb.

Evolution of nitrilases in glucosinolate-containing plants.

Nitrilases, enzymes that catalyze the hydrolysis of organic cyanides, are ubiquitous in the plant kingdom. The typical plant nitrilase is a nitrilase 4 homolog which is involved in the cyanide detoxification pathway. In this pathway, nitrilase 4 converts beta-cyanoalanine, the intermediate product of cyanide detoxification, into asparagine, aspartic acid and ammonia. In the Brassicaceae, a new family of nitrilases has evolved, the nitrilase 1 homologs. These enzymes are not able to use beta-cyanoalanine as a substrate. Instead, they display rather broad substrate specificities and are able to hydrolyze nitriles that result from the decomposition of glucosinolates, the typical secondary metabolites of the Brassicaceae. Here we summarize and discuss data indicating that nitrilase 1 homologs have evolved to function in glucosinolate catabolism.

Evolution of heteromeric nitrilase complexes in Poaceae with new functions in nitrile metabolism.

Members of the nitrilase 4 (NIT4) family of higher plants catalyze the conversion of beta-cyanoalanine to aspartic acid and asparagine, a key step in cyanide detoxification. Grasses (Poaceae) possess two different NIT4 homologs (NIT4A and NIT4B), but none of the recombinant Poaceae enzymes analyzed showed activity with beta-cyanoalanine, whereas protein extracts of the same plants clearly posses this activity. Sorghum bicolor contains three NIT4 isoforms SbNIT4A, SbNIT4B1, and SbNIT4B2. Individually, each isoform does not possess enzymatic activity whereas the heteromeric complexes SbNIT4A/B1 and SbNIT4A/B2 hydrolyze beta-cyanoalanine with high activity. In addition, the SbNIT4A/B2 complex accepts additional substrates, the best being 4-hydroxyphenylacetonitrile. Corresponding NIT4A and NIT4B isoforms from other Poaceae species can functionally complement the sorghum isoforms in these complexes. Site-specific mutagenesis of the active site cysteine residue demonstrates that hydrolysis of beta-cyanoalanine is catalyzed by the NIT4A isoform in both complexes whereas hydrolysis of 4-hydroxyphenylacetonitrile occurs at the NIT4B2 isoform. 4-Hydroxyphenylacetonitrile was shown to be an in vitro breakdown product of the cyanogenic glycoside dhurrin, a main constituent in S. bicolor. The results indicate that the SbNIT4A/B2 heterocomplex plays a key role in an endogenous turnover of dhurrin proceeding via 4-hydroxyphenylacetonitrile and thereby avoiding release of toxic hydrogen cyanide. The operation of this pathway would enable plants to use cyanogenic glycosides as transportable and remobilizable nitrogenous storage compounds. Through combinatorial biochemistry and neofunctionalizations, the small family of nitrilases has gained diverse biological functions in nitrile metabolism.