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: A survey has been made of the occurrence and distribution of three enzymes which metabolize cyanide in a variety of higher plants including both cyanogenic and non-cyanogenic species. The enzymes investigated were beta-cyanoalanine synthase, rhodanese and formamide hydrolyase. beta-Cyanoalanine synthase was found to be present in every higher plant tested whereas rhodanese was found to occur far less commonly in plants. Formamide hydrolyase activity was not detected in any of the higher plants tested.In addition, quantitative analyses have been made of the potential hydrogen cyanide content of each plant investigated. A general trend was apparent between the hydrogen cyanide potential and cyanide metabolizing activity, in that the higher the hydrogen cyanide potential, in general, the higher the cyanide metabolizing activity.Plant physiology 07/1980; 65(6):1199-202. · 6.56 Impact Factor
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ABSTRACT: b-Cyano-alanine synthase (CAS; EC 188.8.131.52) plays an important role in cyanide metabolism in plants. Although the enzymatic activity of b-cyano-Ala synthase has been detected in a variety of plants, no cDNA or gene has been identified so far. We hypothesized that the mitochondrial cysteine synthase (CS; EC 184.108.40.206) isoform, Bsas3, could actually be identical to CAS in spinach (Spinacia oleracea) and Arabidopsis. An Arabidopsis expressed sequence tag database was searched for putative Bsas3 homologs and four new CS-like isoforms, ARAth;Bsas1;1, ARAth;Bsas3;1, ARAth;Bsas4;1, and ARAth;Bsas4;2, were identified in the process. ARAth;Bsas3;1 protein was homologous to the mitochondrial SPIol;Bsas3;1 isoform from spinach, whereas ARAth;Bsas4;1 and ARAth;Bsas4;2 proteins defined a new class within the CS-like proteins family. In contrast to spinach SPIol;Bsas1;1 and SPIol;Bsas2;1 recombinant proteins, spinach SPIol;Bsas3;1 and Arabidopsis ARAth; Bsas3;1 recombinant proteins exhibited preferred substrate specificities for the CAS reaction rather than for the CS reaction, which identified these Bsas3 isoforms as CAS. Immunoblot studies supported this conclusion. This is the first report of the identification of CAS synthase-encoding cDNAs in a living organism. A new nomenclature for CS-like proteins in plants is also proposed.Plant physiology 08/2000; 123(3). · 6.56 Impact Factor
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ABSTRACT: Beta-cyano-L-alanine synthase [L-cysteine hydrogen-sulfide-lyase (adding HCN), EC 220.127.116.11] was purified about 4000-fold from blue lupine seedlings. The enzyme was homoegeneous on gel electrophoresis and free of contamination by other pyridoxal-P-dependent lyases. The enzyme has a molecular weight of 52,000 and contains 1 mole of pyridoxal-P per mole of protein; its isoelectric point is situated at pH 4.7. Its absorption spectrum has two maxima, at 280 and 410 nm. L-Cysteine is the natural primary (amino acid) substrate; beta-chloro- and beta-thiocyano can serve (with considerably lower affinity) instead of cyanide as cosubstrates for cyanoalanine synthase. The synthase is refractory to DL-cycloserine and D-penicillamine, potent inhibitors of many pyridoxal-P-dependent enzymes. Cyanoalanine synthase catalyzes slow isotopic alpha-H exchange in cysteine and in end-product amino acids; the rates of alpha-H exchange in nonreacted (excess) cysteine are markedly increased in the presence of an adequate cosubstrate; no exchange is observed of H atoms in beta-position.Proceedings of the National Academy of Sciences 05/1975; 72(4):1617-21. · 9.74 Impact Factor