Abstract
Reactive carbonyls, especially α,β-unsaturated carbonyls produced through lipid peroxidation, damage biomolecules such as proteins and nucleotides; elimination of these carbonyls is therefore essential for maintaining cellular homeostasis. In this study, we focused on an NADPH-dependent detoxification of reactive carbonyls in plants and explored the enzyme system involved in this detoxification process. Using acrolein (CH(2) = CHCHO) as a model α,β-unsaturated carbonyl, we purified a predominant NADPH-dependent acrolein-reducing enzyme from cucumber leaves, and we identified the enzyme as an alkenal/one oxidoreductase (AOR) catalyzing reduction of an α,β-unsaturated bond. Cloning of cDNA encoding AORs revealed that cucumber contains two distinct AORs, chloroplastic AOR and cytosolic AOR. Homologs of cucumber AORs were found among various plant species, including Arabidopsis, and we confirmed that a homolog of Arabidopsis (At1g23740) also had AOR activity. Phylogenetic analysis showed that these AORs belong to a novel class of AORs. They preferentially reduced α,β-unsaturated ketones rather than α,β-unsaturated aldehydes. Furthermore, we selected candidates of other classes of enzymes involved in NADPH-dependent reduction of carbonyls based on the bioinformatic information, and we found that an aldo-keto reductase (At2g37770) and aldehyde reductases (At1g54870 and At3g04000) were implicated in the reduction of an aldehyde group of saturated aldehydes and methylglyoxal as well as α,β-unsaturated aldehydes in chloroplasts. These results suggest that different classes of NADPH-dependent reductases cooperatively contribute to the detoxification of reactive carbonyls.
Highlights
When plants are subjected to abiotic and/or biotic stresses, oxidative stress often results; this leads to the production of reactive oxygen species, which damage biomolecules such as proteins and lipids
We selected candidates of other classes of enzymes involved in NADPH-dependent reduction of carbonyls based on the bioinformatic information, and we found that an aldo-keto reductase (At2g37770) and aldehyde reductases (At1g54870 and At3g04000) were implicated in the reduction of an aldehyde group of saturated aldehydes and methylglyoxal as well as ␣,-unsaturated aldehydes in chloroplasts
Our results show that one aldo-keto reductase (AKR) and two short-chain dehydrogenase/reductase (SDR) catalyze the NADPH-dependent reduction of aldehyde groups in both saturated and unsaturated aldehyde compounds in chloroplasts; the SDRs were identified as aldehyde reductases (ADRs) based on their catalyzing reaction
Summary
Chemicals—Acrolein, crotonaldehyde, 3-buten-2-one, (E)-2-pentenal, allyl alcohol, and 2-methyl-2-butenal were purchased from Tokyo Kasei Kogyo (Tokyo, Japan). 1-Penten-3one, 3-penten-2-one, 3-methyl-2-butenal, 2-cyclohexen-1one, and methylglyoxal were purchased from Sigma. Purification of Acrolein-reducing Enzyme—Cucumber leaves (100 g) were frozen with liquid nitrogen, and proteins were extracted with 5 volumes of 50 mM HEPES-NaOH, pH 7.0, containing 1 mM dithiothreitol (DTT) and 0.1 mM EDTA. Proteins precipitating between 35 and 55% ammonium sulfate saturation were dissolved in a minimum volume of 50 mM HEPES-NaOH, pH 7.0, containing 1 mM DTT (buffer A) and dialyzed against buffer A. After an equal volume of 60% ammonium sulfate saturated in 50 mM K-PB, pH 7.0, containing 1 mM 2-mercaptoethanol was added to the active fraction, the solution was applied to a phenyl-Sepharose column (1.5 ϫ 8 cm, GE Healthcare) equilibrated with 30% ammonium sulfate saturated in 50 mM K-PB, pH 7.0, containing 1 mM 2-mercaptoethanol and eluted with a linear gradient of 30 to 0% ammonium sulfate (total 100 ml). Arabidopsis Genome Initiative locus identifiers for the genes mentioned in this article are as follows: AtAOR, At1g23740; AtChlADR, At1g54870 and At3g04000; AtCytADR, At2g24190, and At3g61220; AtChlAKR, At2g37770; AtAER, At5g16970
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