Purification, Characterization, and Cloning of α-Hydroxynitrile Lyase from Cassava (Manihot esculenta Crantz)

Hughes, Jane; Carvalho, Frances; Hughes, Monica A.

doi:10.1006/abbi.1994.1267

Cited by 122 publications

(86 citation statements)

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“…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, 1994White et al, , 1998White 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). Harvesting operations generally cause tissue disruption, which allows linamarin and linamarase to come into contact, initiating cyanogenesis.…”

Section: Discussionmentioning

confidence: 99%

Extending Cassava Root Shelf Life via Reduction of Reactive Oxygen Species Production

Zidenga

Leyva-Guerrero²,

Moon³

et al. 2012

Plant Physiology

138

168

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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 cyanideinduced oxidative stress as well as PPD control strategies involving inhibition of ROS production or its sequestration.

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Section: Discussionmentioning

confidence: 99%

Extending Cassava Root Shelf Life via Reduction of Reactive Oxygen Species Production

Zidenga

Leyva-Guerrero²,

Moon³

et al. 2012

Plant Physiology

138

168

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“…(Rosaceae) [9] but accepted as well by H. brasiliensis HNL. The specific activity was measured by following the formation of Fig.…”

Section: Substrate Specificity Experiments With Racemic Mandelonitrilementioning

confidence: 99%

Potential active‐site residues in polyneuridine aldehyde esterase, a central enzyme of indole alkaloid biosynthesis, by modelling and site‐directed mutagenesis

Mattern-Dogru¹,

Ma²,

Hartmann³

et al. 2002

European Journal of Biochemistry

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In the biosynthesis of the antiarrhythmic alkaloid ajmaline, polyneuridine aldehyde esterase (PNAE) catalyses a central reaction by transforming polyneuridine aldehyde into epivellosimine, which is the immediate precursor for the synthesis of the ajmalane skeleton. The PNAE cDNA was previously heterologously expressed in E. coli. Sequence alignments indicated that PNAE has a 43% identity to a hydroxynitrile lyase from Hevea brasiliensis, which is a member of the a/b hydrolase superfamily. The catalytic triad, which is typical for this family, is conserved. By sitedirected mutagenesis, the members of the catalytic triad were identified. For further detection of the active residues, a model of PNAE was constructed based on the X-ray crystallographic structure of hydroxynitrile lyase. The potential active site residues were selected on this model, and were mutated in order to better understand the relationship of PNAE with the a/b hydrolases, and as well its mechanism of action. The results showed that PNAE is a novel member of the a/b hydrolase enzyme superfamily.Keywords: polyneuridine aldehyde esterase; active-site residues; site-directed mutagenesis; modelling; a/b hydrolase enzyme superfamily.Polyneuridine aldehyde esterase (PNAE, EC 3.1.1) is a central enzyme in a 10-step biosynthetic pathway expressed in the medicinal plant Rauvolfia serpentina Benth. ex Kurz. The pathway delivers the antiarrhythmic monoterpenoid indole alkaloid ajmaline [1,2]. From the enzymatic point of view, this is presently one of the most detailed investigated examples of a pathway leading to a natural product. Reactions of this complex biosynthetic sequence are catalyzed by membrane-bound and soluble enzymes such as cytochrome P450-dependent hydroxylases, a synthase, several reductases, a methyltransferase and a set of hydrolases including a b-glucosidase, an acetylesterase and the herein described methylesterase PNAE [3]. These enzymes exhibit a high degree of substrate specificity, PNAE being the most specific. PNAE is located in the middle of the biosynthetic chain starting from tryptamine and secologanin and catalyses the conversion of polyneuridine aldehyde into the next stable pathway intermediate, epivellosimine (Fig. 1). The enzyme has been detected in and partially characterized from cell suspension cultures of R. serpentina [4,5]. PNAE has also been highly enriched from cultured plant cells and the cDNA was recently functionally expressed in Escherichia coli [6]. Sequence alignment studies showed highest homologies to an esterase involved in pathogen defence in rice [7] that hydrolyses naphthol esters, and to hydroxynitrile lyases (HNLs) from Hevea brasiliensis [8] and Manihot esculenta [9]. The alignment suggested that PNAE is a new member of the a/b hydrolase superfamily that contains the putative catalytic triad serine, aspartic acid and histidine [10].For the present communication, a more detailed characterization of PNAE was performed by testing the influence of various inhibitors, HNL substrates and by structure elucid...

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“…Depending on their stereo preferences for asymmetric cyanohydrin synthesis, HNLs are often divided into two classes: (S)-HNL and (R)-HNL. (S)-HNLs are found and investigated in Ximenia americana (XaHNL), 3) Manihot esculenta (MeHNL), 4) Hevea brasiliensis (HbHNL), 5) and Sorghum bicolor (SbHNL). 6) (R)-HNLs are Phlebodium aureum (PhaHNL), 7) Linum usitatissimum (LuHNL), 8,9) Arabidopsis thaliana (AtHNL), 10) and Pr.…”

mentioning

confidence: 99%

Characterization of a New (R)-Hydroxynitrile Lyase from the Japanese ApricotPrunus mumeand cDNA Cloning and Secretory Expression of One of the Isozymes inPichia pastoris

Fukuta

Nanda

Kato

et al. 2011

Bioscience, Biotechnology, and Biochemistry

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Purification, Characterization, and Cloning of α-Hydroxynitrile Lyase from Cassava (Manihot esculenta Crantz)

Cited by 122 publications

References 0 publications

Extending Cassava Root Shelf Life via Reduction of Reactive Oxygen Species Production

Extending Cassava Root Shelf Life via Reduction of Reactive Oxygen Species Production

Potential active‐site residues in polyneuridine aldehyde esterase, a central enzyme of indole alkaloid biosynthesis, by modelling and site‐directed mutagenesis

Characterization of a New (R)-Hydroxynitrile Lyase from the Japanese ApricotPrunus mumeand cDNA Cloning and Secretory Expression of One of the Isozymes inPichia pastoris

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