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Isolation and characterization of AaAER, a novel double bond reductase from Artemisia annua

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Abstract

We isolated a 2-alkenal reductase (AaAER) gene from Artemisia annua L. cDNA library through homologous cloning strategy. Enzymatic properties of AaAER, including substrate selectivity, enzyme kinetics, and key factors affecting enzyme activity, were characterized in vitro. We found that AaAER mainly functions in catalyzing the reduction of adjacent straight chain “C = C” double bond in the carbonyl group. This suggests the possibility of its participation in downstream events of the HPL (hydroperoxide lyase) pathway and contribution to cell detoxification in plants. Although, AaAER shares some substrates with AaDbr 1 and AaDbr 2, which are involved in artemisinin biosynthesis, AaAER was unable to reduce the adjacent double bond of carbonyl group in artemisinic aldehyde. We also analyzed the possible causes for highly homologous reductases with different types of substrates and non-homologous enzymes sharing the same substrate. The identification of a novel double bond reductase in A. annua provides an opportunity to explore the mechanism underlying the detoxification of aldehyde ketone molecules, which are generated by HPL downstream pathway and the redox process in the metabolic pathway of artemisinin synthesis.

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Abbreviations

MeJA:

Methyl jasmonate

SA:

Salicylic acid

RACE:

Rapid amplification of cDNA ends

AaAER:

2-alkenal reductase of Artemisia annua

Dbr1:

Double bond reductase 1

Dbr2:

Artemisinic aldehyde Δ11(13) reductase

MEGA:

Molecular evolutionary genetics analysis

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Authors and Affiliations

  1. Shanghai Chenshan Plant Science Research Center, Chinese Academy of Sciences, Shanghai Chenshan Botanical Garden, 3888 Chenhua Road, Songjiang, Shanghai, 201602, People’s Republic of China

    Yu-Kun Wei

  2. Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Feng Lin Road, Shanghai, 200032, People’s Republic of China

    Jian-Xu Li, Wen-Li Hu, Chang-Qing Yang & Ling-Jian Wang

Authors
  1. Yu-Kun Wei
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  2. Jian-Xu Li
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  3. Wen-Li Hu
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  4. Chang-Qing Yang
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  5. Ling-Jian Wang
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Correspondence to Yu-Kun Wei.

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Table S1

The primers used in this study (DOC 32 kb)

Fig. S1

The alignment of amino acid sequences of AaAER and its homologs. The boxed AXXGXXG is NADP + binding site. Three glycines indicated with arrowheads are key sites of the active enzyme with high-level structure. (GIF 189 kb)

High resolution image (TIFF 256 kb)

Fig. S2

Inducible expression and purification of AaAER. (a) In vitro Inducible expression and purification of AaAER protein. Lane 1, in vitro total AaAER protein; lane 2, empty vector; lane 3, 10 mM imidazol elution; lane 4–5, 20 mM imidazol elution; lane 6–8 250 mM imidazol elution; Lane 9, protein marker. (b) Western blotting data further identified target proteins. Data showed the protein size of the target is around 55 kD. Lane 1, protein target; lane 2, protein marker. (GIF 91 kb)

High resolution image (TIFF 1722 kb)

Fig. S3

GC-MS analyses of the reaction between AaAER and 2-nonenal. Reduction activity of AaAER was identified in reaction systems with inactive proteins and without NADPH. Product was identified through GC-MS chromatogram compared to standard. a, Chromatogram of product (2-nonanal). b, Chromatogram of standard (2-nonanal). (GIF 219 kb)

High resolution image (TIFF 3063 kb)

Fig. S4

GC-MS analyses of the reaction between AaAER and 3-nonen-2-one. Reduction activity of AaAER was identified in reaction systems with inactived proteins. Product was identified through GC-MS chromatogram compared to standard. a, Chromatogram of product (2-nonanone). b, Chromatogram of standard (2-nonanone). (GIF 161 kb)

High resolution image (TIFF 3333 kb)

Fig. S5

GC-MS analyses of the reaction between AaAER and cinnamaldehyde. Reduction activity of AaAER was identified in reaction systems with inactivate proteins. Product was identified through GC-MS chromatogram compared to standard. a, Chromatogram of product (hydrocinnamaldehyde). b, Chromatogram of standard (hydrocinnamaldehyde). (GIF 172 kb)

High resolution image (TIFF 3517 kb)

Fig. S6

GC-MS analyses of the reaction between AaAER and trans-2, cis-6-nonadienal. Product was identified through GC-MS chromatogram compared to standard. a, Chromatogram of product (6-nonenal). b, Chromatogram of standard (6-nonenal). (GIF 198 kb)

High resolution image (TIFF 3249 kb)

Fig. S7

Effect of temperature on AaAER activity. Reactions with 3-nonen-2-one as a substrate were carried out in HEPES buffer at pH 6.0. The curve of reaction rate against the temperature. (GIF 13 kb)

High resolution image (TIFF 16899 kb)

Fig. S8

Effects of pH on AaAER activity. Reactions were carried out in HEPES buffer at 37 °C. The curve of reaction rate against pH. (GIF 12 kb)

High resolution image (TIFF 16437 kb)

Fig. S9

Effects of metal ions on AaAER activity. Comparison of reaction rate with different metal ions. HEPES buffer without metal ions was used as a control. Reactions with 3-nonen-2-one as a substrate were carried out at 37 °C and pH 6.0. The difference in the average reaction rate of different metal ions was evaluated by Dunnett’s Multiple Comparison Test. ** and *** indicate different degrees of difference compared to controls. (GIF 28 kb)

High resolution image (TIFF 15612 kb)

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Wei, YK., Li, JX., Hu, WL. et al. Isolation and characterization of AaAER, a novel double bond reductase from Artemisia annua . J. Plant Biochem. Biotechnol. 24, 331–337 (2015). https://doi.org/10.1007/s13562-014-0278-2

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  • DOI: https://doi.org/10.1007/s13562-014-0278-2

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