Purification, Characterization, and Cloning of α-Hydroxynitrile Lyase from Cassava (Manihot esculenta Crantz)
alpha-Hydroxynitrile lyase (HNL, acetone cyanohydrin lyase, EC 184.108.40.206) was purified to homogeneity from young leaves of the cyanogenic tropical crop plant cassava (Manihot esculenta Crantz). The purified protein is a homo-trimer with a subunit relative molecular mass of 28,500 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The active protein is not glycosylated and does not contain a flavin group. HNL exhibits complex kinetics which vary according to substrate concentration and may be related to aggregation of the enzyme. HNL activity against two natural substrates, acetone cyanohydrin and 2-butanone cyanohydrin, and one nonphysiological substrate, 2-pentanone cyanohydrin, was demonstrated. N-terminal and internal peptide sequences, obtained from HNL digested with the endoproteinase Glu-C, were used to design degenerate oligonucleotide primers for polymerase chain reaction with single-strand cDNA, using purified mRNA from cotyledons as template. The resulting DNA fragment was used to probe a cassava cotyledon cDNA library. Four cDNA clones were isolated, sequenced, and shown to contain derived amino acid sequences identical to those obtained from the purified protein.
Available from: Sukhuman Whankaew
- "Specific primers were designed using the PrimerQuest software (http://sg.idtdna.com/primerquest/Home/Index). The sequences of the target genes, LNM and HNL, sourced from previous publications (Hughes et al., 1992, 1994) were also used for primer design. The criterion for amplified products ranged from 100 to 300 bp with a Tm of 60 ± 5°C. "
Available from: Hangsik Moon
- "The resulting acetone cyanohydrin is unstable and degrades spontaneously at pH levels higher than 5.0 or temperatures greater than 35°C, or enzymatically by hydroxynitrile lyase in leaves, releasing hydrogen cyanide and acetone (Cutler and Conn, 1981; Hughes et al., 1994; White et al., 1994, 1998; White and Sayre, 1995). However, cyanide release generally does not happen in intact cells because linamarin is localized in the vacuole, while the deglycosylase, linamarase, is localized in the cell wall and in laticifers (Mkpong et al., 1990; Hughes et al., 1994; McMahon et al., 1995). "
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ABSTRACT: One of the major constraints facing the large-scale production of cassava (Manihot esculenta) roots is the rapid postharvest physiological deterioration (PPD) that occurs within 72 h following harvest. One of the earliest recognized biochemical events during the initiation of PPD is a rapid burst of reactive oxygen species (ROS) accumulation. We have investigated the source of this oxidative burst to identify possible strategies to limit its extent and to extend cassava root shelf life. We provide evidence for a causal link between cyanogenesis and the onset of the oxidative burst that triggers PPD. By measuring ROS accumulation in transgenic low-cyanogen plants with and without cyanide complementation, we show that PPD is cyanide dependent, presumably resulting from a cyanide-dependent inhibition of respiration. To reduce cyanide-dependent ROS production in cassava root mitochondria, we generated transgenic plants expressing a codon-optimized Arabidopsis (Arabidopsis thaliana) mitochondrial alternative oxidase gene (AOX1A). Unlike cytochrome c oxidase, AOX is cyanide insensitive. Transgenic plants overexpressing AOX exhibited over a 10-fold reduction in ROS accumulation compared with wild-type plants. The reduction in ROS accumulation was associated with a delayed onset of PPD by 14 to 21 d after harvest of greenhouse-grown plants. The delay in PPD in transgenic plants was also observed under field conditions, but with a root biomass yield loss in the highest AOX-expressing lines. These data reveal a mechanism for PPD in cassava based on cyanide-induced oxidative stress as well as PPD control strategies involving inhibition of ROS production or its sequestration.
Available from: Deepak Ganjewala
- "UDP-glucose:p-hydroxymandelo- nitrile-O-glucosyltransferase Sorghum bicolor AAF17077 492 UDPGT Jones et al. 1999 A-hydroxynitrile lyase Manihot esculenta CAA82334 AAV52632 CAA11219 CAA11428 258 258 258 158 Abhydrolase_1 HNL HNL4 HNL 24 Hughes et al. 1994 Wang et al. 2004 (S)-hydroxynitrile lyase Hevea brasiliensis AAC49184 257 Hnl Hasslacher et al. 1996 Hydoxynitrile lyase Prunus dulcis AAL11514 IJU2_B 563 536 hnl1 hnl1 Dreveny et al. 2001 (R)-(+)-mandelonitrile lyase Prunus serotina P52707 573 MDL3 Hu and Poulton 1999 Rhodanese Triticum aestivum AAK64575 307 TST Niu et al. 2002 Rhodanese "
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ABSTRACT: A number of species of plants produce repertoire of cyanogenic glycosides via a common biosynthetic scheme. Cyanogenic glycosides play pivotal roles in organization of chemical defense system in plants and in plant–insect interactions. Several commercial crop plants such as sorghum (Sorghum bicolor), cassava (Manihot esculenta) and barley (Hordium vulgare) are cyanogenic and accumulate significant amounts of cyanogenic glycosides. The study of biosynthesis of dhurrin in sorghum has underpinned several early breakthroughs in cyanogenic glycoside researches. Despite great deal of structural diversity in cyanogenic glycosides, almost all of them are believed to be derived from only six different amino acids L-valine, L-isoleucine, L-leucine, L-phenylalanine, or L-tyrosine and cyclopentenyl-glycine (a non protein amino acid). Our knowledge about biosynthesis of cyanogenic glycosides and molecular regulatory processes underlying their biosynthesis has been increased impressively in the past few years. The rapid identification, characterization and cloning of genes encoding enzymes of the cyanogenic glycoside biosynthetic and catabolic pathways from several plants has greatly facilitated our understanding of cyanogenic glycosides biosynthesis and regulation. Today it is known that enzymes of cyanogenic glycoside biosynthetic pathway in sorghum are organized as metabolon most likely to those of other secondary metabolic pathways. Knowledge of state of art of biosynthesis and regulation of cyanogenic glycosides made possible the metabolic engineering of these pathways resulting in development of transgenics of cassava, tobacco, lotus and Arabidopsis with manipulated cyanogenic glycosides content. Simultaneously, many new developments have been witnessed in methods/techniques/ procedures for detection of cyanogenic glycosides in plant samples, foods and foodstuffs. The present review sequentially discusses all of these issues with updated information gathered from the published reports on cyanogenic glycosides.
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