A soluble beta-cyanoalanine synthase from the gut of the variegated grasshopper Zonocerus variegatus (L.).
ABSTRACT Beta-cyanoalanine synthase (beta-cyano-l-alanine synthase; l-cysteine: hydrogen sulphide lyase (adding hydrogen cyanide (HCN)); EC 4. 4.1.9) was purified from the cytosolic fraction of the gut of grasshopper Zonocerus variegatus (L.) by ion-exchange chromatography on DEAE-Cellulose and gel filtration on Sephadex G-100 columns. The crude enzyme had a specific activity of 2.16nmol H2S/min/mg. A purified enzyme with a specific activity, which was seventeen times higher than that of the crude extract, was obtained. A molecular weight of about 55.23+/-1.00Kd was estimated from its elution volume on Sephadex G-100. The fraction when subjected to sodium dodecyl sulphate-polyacrylamide elel electrophoresis revealed the presence of a protein band with Mr of 23.25+/-0.25Kd. The enzyme exhibited Michaelis-Menten kinetics having Km of 0.38mM for l-cysteine and Km of 6.25mM for cyanide. The optimum temperature and pH for activity were determined to be at 30 degrees C and pH 9.0, respectively. This enzyme might be responsible for the ability to detoxify cyanide in this insect pest and hence its tolerance of the cyanogenic cassava plant. Biophysical, biochemical and kinetic properties of this enzyme, which will reveal how this ability can possibly be compromised by enzyme inhibition, may lead, in the long term, to the potential use of this enzyme as drug target for pest control.
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ABSTRACT: O-acetylserine(thiol)lyases (OAS-TLs) play a pivotal role in sulfur assimilation pathway incorporating sulfide into aminoacids in microorganisms and plants, however, these enzymes have not been found in the animal kingdom. Interestingly, the genome of the roundworm Caenorhabditis elegans contains three expressed genes predicted to encode OAS-TL orthologs (cysl-1 - cysl-3), and a related pseudogene (cysl-4); these genes play different roles in resistance to hypoxia, hydrogen sulfide and cyanide. To get insight into the underlying molecular mechanisms we purified the three recombinant worm OAS-TL proteins, and we determined their enzymatic activities, substrate binding affinities, quaternary structures and the conformations of their active site shapes. We show that the nematode OAS-TL orthologs can bind O-acetylserine and catalyze the canonical reaction although this ligand may more likely serve as a competitive inhibitor to natural substrates instead of being a substrate for sulfur assimilation. In addition, we propose that S-sulfocysteine may be a novel endogenous substrate for these proteins. However, we observed that the three OAS-TL proteins are conformationally different and exhibit distinct substrate specificity. Based on the available evidences we propose the following model: CYSL-1 interacts with EGL-9 and activates HIF-1 that upregulates expression of genes detoxifying sulfide and cyanide, the CYSL-2 acts as a cyanoalanine synthase in the cyanide detoxification pathway and simultaneously produces hydrogen sulfide, while the role of CYSL-3 remains unclear although it exhibits sulfhydrylase activity in vitro. All these data indicate that C. elegans OAS-TL paralogs have distinct cellular functions and may play different roles in maintaining hydrogen sulfide homeostasis.Biochimica et Biophysica Acta 10/2013; · 4.66 Impact Factor
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ABSTRACT: While most dicot plants produce little ethylene in their vegetative stage, many monocots such as rice liberate a relatively large amount of ethylene with cyanide as a co-product in their seedling stage when etiolated. One of the known functions of beta-cyanoalanine synthase (CAS) is to detoxify the co-product cyanide during ethylene biosynthesis in higher plants. Based on a tryptic peptide sequence obtained from a partially purified CAS activity protein preparation in etiolated rice seedlings, the full-length putative rice CAS-encoding cDNA sequence (OsCAS), which is homologous to those O-acetylserine sulphydrylase (OASS) genes, was cloned. Unlike most of the CAS genes reported from dicots, the transcription of OsCAS is promoted by auxins but suppressed by ethylene. To address the function and the subcellular localization of this gene product in planta, a binary vector construct consisting of this gene appended with a yellow fluorescent protein-encoding sequence was employed to transform Arabidopsis. Specific activities on CAS and OASS of the purified recombinant protein from transgenic Arabidopsis were 181.04 micromol H(2)S mg(-1) protein min(-1) and 0.92 micromol Cys mg(-1) protein min(-1), respectively, indicating that OsCAS favours CAS activity. The subcellular localization of OsCAS was found mostly in the mitochondria by immunogold electron-microscopy. Chemical cross-linking and in-gel assay on a heterodimer composed of functional and non-functional mutants in a yeast expression system on OsCAS suggested that OsCAS functions as a homodimer, similar to that of OASS. Despite the structural similarity of OsCAS with OASS, it has also been confirmed that OsCAS could not interact with serine-acetyltransferase, indicating that OsCAS mainly functions in cyanide detoxification.Journal of Experimental Botany 02/2009; 60(3):993-1008. · 5.24 Impact Factor
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ABSTRACT: The grasshopper (Zonocerus variegatus) has been consumed for centuries in Africa, Asia and some other parts of the world. It has a superior nutritional content compared to other animal protein but has received little attention globally for various reasons. As the world strives to overcome global hunger and malnutrion, especially in underdeveloped and developing countries, this insect shows tremendous potential as an alternative rich protein source with its resilience and abundance in nature. More scientific research is needed to explore the potential of this insect to help alleviate the food needs of the world's current 7 billion people. INTRODUCTION Entomologists have described edible insects as "microlivestock"(Lyon,1991). Although entomophagy (the human consumption of insects) has not received significant attention in western Literatures, inspite of the superior nutritional content of edible insects compared to other animals (Lyon, 1991; Iligner and Nel, 2000) this has been attributed to nothing more than customs and prejudice (Owen, 1973). This organism is highly abundant in nature and varies in abundance depending on the type of vegetation cover in a region with forests with Chromolaena odorata much more favoured (Kekeunou et al., 2007). It is the dominant species among grasshoppers in many farmlands in Nigeria and elsewhere (Oku et al., 2011). Too much attention has been focused on the destruction of this potential alternative food source rather than on its preservation and growth (Müller, De Groote, Gbongboui, and Langewald, 2002; Ogunlabi and Agboola, 2007). There is a long history of insects' consumption in Africa and Asia (Sutton, 1988; Mian, 2003; Agbidye et al., 2009) and we will use Nigeria as a case study. Several studies in Nigeria have shown that the practice of entomophagy has contributed significantly to the reduction of protein deficiencies in the country (Ene, 1963; Ashiru, 1988; Fasoranti and Ajiboye, 1993; Banjo et al 2006; Omotosho, 2006). A number of insects or their products were used as food in many parts of Nigeria (Ene, 1963). The potentials of Zonocerus variegatus Insects are widespread on earth (Demirel and Cranshaw, 2006) and adapt to all manner of climate, weather and altitude conditions (Idris et al., 2002; Riedel et al., 2008). The most widely consumed insect species are grasshoppers (Burnie, 2007). Many modern entomophagers contend that insects should be the food of the future because they are nutritionally superior to many other meat protein sources, such as beef and chicken. In addition, insects are abundant; they constitute about 75% of known species of animals (Yoloye, 1988). With about 1500 identified edible species (Dufour, 1987; Smith, 1999), some insects are eaten as larvae, others in their adult stage (Wikipedia, 2010). In deserts or sub-Saharan environments and developing countries where food and water is scarce, and malnutrition has been reported to be very high (Joosten and Hulst, 2011; Norman, Pichard, Lochs, and Pirlich, 2008; Saunders, Smith, & Stroud, 2011) these insects can be an important food stable source (Kho, 2002). The northeast region of Nigeria is the second largest livestock producer in Nigeria (Majiayagbe and Lamorde, 1997), but very little has been done or documented about the rich "microlivestock" population of this region and the high cost of animal protein, which is beyond the reach of the highly impoverished population has reawakened and encouraged entomophagy in this part of the country more than other parts. This renewed interest in entomophagy will also also help to reduce the pest role of Zonocerus variegtus which requires attracticides (Timbilla et al., 2007) to control in Africa and many parts of the world.Continental J. Food Science and Technology. 01/2012; 6(2):8-12.