A soluble beta-cyanoalanine synthase from the gut of the variegated grasshopper Zonocerus variegatus (L.).

Department of Biochemistry, Olabisi Onabanjo University Remo Campus, Ikenne, Ogun State, Nigeria.
Insect Biochemistry and Molecular Biology (Impact Factor: 3.42). 02/2007; 37(1):72-9. DOI: 10.1016/j.ibmb.2006.10.003
Source: PubMed

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.

  • [Show abstract] [Hide abstract]
    ABSTRACT: The purification and characterization of rhodanese, an enzyme that catalyses the detoxification of cyanide, from the gut of the variegated grasshopper (Zonocerus variegatus L.) were carried out to understand the biochemical basis of the survival of this grasshopper living on cyanogenic plants such as cassava. All experiments, including enzyme assay, were carried out at room temperature and all buffers contained 10 mm sodium thiosulphate to stabilize the enzyme. Grasshoppers were caught alive from a cassava farm within the locality and kept frozen until analysis. Each grasshopper was dissected and the gut was removed quickly. Approximately 102 g of the gut were homogenized in three volumes of 0.1 m acetate glycine buffer (pH 7.8) containing ɛ-amino-n-caproic acid. The supernatant was collected by centrifugation at 12,000 rpm, for 30 min at 40 °C. The enzyme was purified to homogeneity by a combination of procedures such as ammonium sulphate precipitation, ion-exchange chromatography (CM-Sephadex and DEAE-Sephadex), gel filtration (Sephadex G-75) and Agarose-Blue affinity chromatography. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE) and non-SDS–PAGE were used to ascertain the purity of the enzyme. The native and subunit molecular weights of the enzyme were determined by gel filtration on a Bio-Gel P-200 column and SDS–PAGE, respectively. Kinetic parameters were determined by using varying concentrations of one of the substrates at a fixed concentration of the other, and vice versa. Furthermore, the effects of temperature, pH and cations on the activity of the enzyme were investigated. The purified enzyme had a specific activity of 51.7 μmol thiocyanate formed/ml/min/mg protein (U/mg protein) with a yield of about 29%. The apparent molecular weight of the enzyme estimated by Sephadex G-75 gel filtration was 35,400 ± 482 Da and its subunit molecular weight determined by SDS–PAGE was 33,000 ± 212 Da. The K m values of KCN and Na2S2O3 were found to be 29.63 ± 02.87 and 26 ± 03.04 mm, respectively. An optimum pH and temperature of 7.0 and 35 °C, respectively, were obtained for the enzyme. The results of enzyme inhibition showed that the activity of the enzyme was not affected by NH4Cl, MgCl2, CoCl2, CaCl2, MnCl2, NiCl2 and SnCl2, but inhibited by ZnCl2 and BaCl2. In conclusion, these results suggest that the survival of Z. variegatus depends on the presence of the enzyme rhodanese, which shows high activity and has suitable kinetic properties in the gut of the grasshopper that feeds mainly on cassava leaves which are cyanogenic.
    International Journal of Tropical Insect Science 09/2013; 33(03). DOI:10.1017/S1742758413000180
  • [Show abstract] [Hide abstract]
    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; DOI:10.1016/j.bbapap.2013.09.020 · 4.66 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    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.