Plant

Plant Physiol. (1998) 116: 1219–1225

Cyanogenesis in Cassava1
The Role of Hydroxynitrile Lyase in Root Cyanide Production
Wanda L.B. White, Diana I. Arias-Garzon, Jennifer M. McMahon, and Richard T. Sayre* Department of Plant Biology, 1735 Neil Avenue (W.L.B.W., D. I.A.-G., J.M.M.), and Departments of Biochemistry and Plant Biology, 2021 Coffey Road, Ohio State University, Columbus, Ohio43210 (R.T.S.)
All cassava tissues, with the exception of seeds, contain the cyanogenic glycosides linamarin ( 90% total cyanogen) and lotaustralin ( 10% total cyanogen; for review, see McMahon et al., 1995). Leaves have the highest cyanogenic glycoside levels (5.0 g linamarin/kg fresh weight), whereas roots have approximately 20-fold lower linamarin levels. In addition to tissue-specificdifferences, there are cultivardependent differences in root cyanogen levels. Total root linamarin levels range between 100 and 500 mg linamarin/kg fresh weight for low- and high-cyanogenic cultivars, respectively. No cassava cultivars, however, lack cyanogenic glycosides. Cyanogenesis is initiated in cassava when the plant tissue is damaged. Rupture of the vacuole releases linamarin, which is hydrolyzed bylinamarase, a cell wall-associated -glycosidase (McMahon et al., 1995). Hydrolysis of linamarin yields an unstable hydroxynitrile intermediate, acetone cyanohydrin, plus Glc. Acetone cyanohydrin spontaneously decomposes to acetone and HCN at pH 5.0 or temperatures 35°C and can be broken down enzymatically by HNL (Cutler and Conn, 1981; Yemm and Poulton, 1986; Wajant and Mundry, 1993; Wajant etal., 1994; White et al., 1994; White and Sayre, 1995; Zheng and Poulton, 1995; Hasslacher et al., 1996; Wajant and Pfizenmaier, 1996). Various health disorders are associated with the consumption of cassava, which contains residual cyanogens. These disorders include hyperthyroidism, tropical ataxic neuropathy, and konzo (Osuntokun, 1981; Cock, 1985; Tylleskar et al., 1992; Rosling et al., 1993).Cyanide poisoning from high-cyanogenic cassava is typically associated with insufficient consumption of Cys and Met in the diet. Reduced sulfur-containing compounds are substrates for the detoxification of cyanide catalyzed by the enzymes rhodanese and/or -cyanoalanine synthase (Castric et al., 1972; Kakes, 1990; Nambisan, 1993). Until recently, it had been assumed that all of the residual cyanogenpresent in cassava foods was in the form of linamarin. This assumption was based on the observation that acetone cyanohydrin is unstable and that the cyanide generated from acetone cyanohydrin is readily volatilized during food processing. Recently, however, it was demonstrated that the major cyanogen present in some poorly processed cassava roots was acetone cyanohydrin, not linamarin(TylAbbreviation: HNL, hydroxynitrile lyase. 1219

In the cyanogenic crop cassava (Manihot esculenta, Crantz), the final step in cyanide production is the conversion of acetone cyanohydrin, the deglycosylation product of linamarin, to cyanide plus acetone. This process occurs spontaneously at pH greater than 5.0 or enzymatically and is catalyzed by hydroxynitrile lyase (HNL). Recently, it has beendemonstrated that acetone cyanohydrin is present in poorly processed cassava root food products. Since it has generally been assumed that HNL is present in all cassava tissues, we reinvestigated the enzymatic properties and tissue-specific distribution of HNL in cassava. We report the development of a rapid two-step purification protocol for cassava HNL, which yields an enzyme that is catalytically moreefficient than previously reported (Hughes, J., Carvalho, F., and Hughes, M. [1994] Arch Biochem Biophys 311: 496–502). Analyses of the distribution of HNL activity and protein indicate that the accumulation of acetone cyanohydrin in roots is due to the absence of HNL, not to inhibition of the enzyme. Furthermore, the absence of HNL in roots and stems is associated with very low steady-state HNL...