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. 1999 Feb;119(2):621-6.
doi: 10.1104/pp.119.2.621.

Purification and characterization of a NADPH-dependent aldehyde reductase from mung bean that detoxifies eutypine, a toxin from eutypa lata1

S Colrat  1 , A Latche, M Guis, JC Pech, M Bouzayen, J Fallot, JP Roustan
Affiliations

Purification and characterization of a NADPH-dependent aldehyde reductase from mung bean that detoxifies eutypine, a toxin from eutypa lata1

S Colrat et al. Plant Physiol. 1999 Feb.

Abstract

Eutypine (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzaldehyde) is a toxin produced by Eutypa lata, the causal agent of eutypa dieback in the grapevine (Vitis vinifera). Eutypine is enzymatically converted by numerous plant tissues into eutypinol (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzyl alcohol), a metabolite that is nontoxic to grapevine. We report a four-step procedure for the purification to apparent electrophoretic homogeneity of a eutypine-reducing enzyme (ERE) from etiolated mung bean (Vigna radiata) hypocotyls. The purified protein is a monomer of 36 kD, uses NADPH as a cofactor, and exhibits a Km value of 6.3 &mgr;M for eutypine and a high affinity for 3- and 4-nitro-benzaldehyde. The enzyme failed to catalyze the reverse reaction using eutypinol as a substrate. ERE detoxifies eutypine efficiently over a pH range from 6.2 to 7.5. These data strongly suggest that ERE is an aldehyde reductase that could probably be classified into the aldo-keto reductase superfamily. We discuss the possible role of this enzyme in eutypine detoxification.

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Figures

Figure 1
Figure 1
Chromatograms of ERE purification. Elution profiles of proteins (⋄) and ERE activity (♦) from Phenyl Sepharose (A), hydroxylapatite (B), Superose 12 (C), and Mono-Q (D) columns.
Figure 2
Figure 2
Silver-stained SDS-PAGE documenting the progress of purification of ERE. Fractions include: Lane 1, Crude extract; lane 2, (NH4)2SO4; lane 3, Phenyl-Sepharose chromatography; lane 4, hydroxyapatite chromatography; lane 5, Superose 12 chromatography; lane 6, Mono-Q chromatography. The positions of molecular mass markers are indicated on the left in kD.
Figure 3
Figure 3
Correlation of kcat with ς, dependent on the electronic nature of the substituent R, for ERE. a, Variation of log (apparent kcat) with ς for substituted benzaldehydes. b, Variation of log (apparent kcat) with ς for 3-substituted benzaldehydes. c, Variation of log (apparent kcat) with ς for 4-substituted benzaldehydes.
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