Dataset Information

Overexpression and Suppression of Artemisia annua 4-Hydroxy-3-Methylbut-2-enyl Diphosphate Reductase 1 Gene (AaHDR1) Differentially Regulate Artemisinin and Terpenoid Biosynthesis.

ABSTRACT: 4-Hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR) catalyzes the last step of the 2-C-methyl-D-erythritol 4- phosphate (MEP) pathway to synthesize isopentenyl pyrophosphate (IPP) and dimethylallyl diphosphate (DMAPP). To date, little is known regarding effects of an increase or a decrease of a HDR expression on terpenoid and other metabolite profiles in plants. In our study, an Artemisia annua HDR cDNA (namely AaHDR1) was cloned from leaves. Expression profiling showed that it was highly expressed in leaves, roots, stems, and flowers with different levels. Green florescence protein fusion and confocal microscope analyses showed that AaHDR1 was localized in chloroplasts. The overexpression of AaHDR1 increased contents of artemisinin, arteannuin B and other sesquiterpenes, and multiple monoterpenes. By contrast, the suppression of AaHDR1 by anti-sense led to opposite results. In addition, an untargeted metabolic profiling showed that the overexpression and suppression altered non-polar metabolite profiles. In conclusion, the overexpression and suppression of AaHDR1 protein level in plastids differentially affect artemisinin and other terpenoid biosynthesis, and alter non-polar metabolite profiles of A. annua. Particularly, its overexpression leading to the increase of artemisinin production is informative to future metabolic engineering of this antimalarial medicine.

SUBMITTER: Ma D

PROVIDER: S-EPMC5281613 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Publications

Overexpression and Suppression of Artemisia annua 4-Hydroxy-3-Methylbut-2-enyl Diphosphate Reductase 1 Gene (AaHDR1) Differentially Regulate Artemisinin and Terpenoid Biosynthesis.

Ma Dongming D Li Gui G Zhu Yue Y Xie De-Yu DY

Frontiers in plant science 20170131

4-Hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR) catalyzes the last step of the 2-C-methyl-D-erythritol 4- phosphate (MEP) pathway to synthesize isopentenyl pyrophosphate (IPP) and dimethylallyl diphosphate (DMAPP). To date, little is known regarding effects of an increase or a decrease of a HDR expression on terpenoid and other metabolite profiles in plants. In our study, an Artemisia annua HDR cDNA (namely AaHDR1) was cloned from leaves. Expression profiling showed ...[more]

PMID: 28197158

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Project description:(E)-4-Hydroxy-3-methylbut-2-enyl diphosphate reductase (IspH or LytB) catalyzes the terminal step of the MEP/DOXP pathway where it converts (E)-4-hydroxy-3-methylbut-2-enyl diphosphate (HMBPP) into the two products, isopentenyl diphosphate and dimethylallyl diphosphate. The reaction involves the reductive elimination of the C4 hydroxyl group, using a total of two electrons. Here we show that the active form of IspH contains a [4Fe-4S] cluster and not the [3Fe-4S] form. Our studies show that the cluster is the direct electron source for the reaction and that a reaction intermediate is bound directly to the cluster. This active form has been trapped in a state, dubbed FeS(A), that was detected by electron paramagnetic resonance (EPR) spectroscopy when one-electron-reduced IspH was incubated with HMBPP. In addition, three mutants of IspH have been prepared and studied, His42, His124, and Glu126 (Aquifex aeolicus numbering), with particular attention paid to the effects on the cluster properties and possible reaction intermediates. None of the mutants significantly affected the properties of the [4Fe-4S](+) cluster, but different effects were observed when one-electron-reduced forms were incubated with HMBPP. Replacing His42 led to an increased K(M) value and a much lower catalytic efficiency, confirming the role of this residue in substrate binding. Replacing the His124 also resulted in a lower catalytic efficiency. In this case, however, the enzyme showed the loss of the [4Fe-4S](+) EPR signal upon addition of HMBPP without the subsequent formation of the FeS(A) signal. Instead, a radical-type signal was observed in some of the samples, indicating that this residue plays a role in the correct positioning of the substrate. The incorrect orientation in the mutant leads to the formation of substrate-based radicals instead of the cluster-bound intermediate complex FeS(A). Replacing the Glu126 also resulted in a lower catalytic efficiency, with yet a third type of EPR signal being detected upon incubation with HMBPP. (31)P and (2)H ENDOR measurements of the FeS(A) species incubated with regular and (2)H-C4-labeled HMBPP reveal that the substrate binds to the enzyme in the proximity of the active-site cluster with C4 adjacent to the site of linkage between the FeS cluster and HMBPP. Comparison of the spectroscopic properties of this intermediate to those of intermediates detected in (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase and ferredoxin:thioredoxin reductase suggests that HMBPP binds to the FeS cluster via its hydroxyl group instead of a side-on binding as previously proposed for the species detected in the inactive Glu126 variant. Consequences for the IspH reaction mechanism are discussed.

Dataset Information

Overexpression and Suppression of Artemisia annua 4-Hydroxy-3-Methylbut-2-enyl Diphosphate Reductase 1 Gene (AaHDR1) Differentially Regulate Artemisinin and Terpenoid Biosynthesis.

Publications

Overexpression and Suppression of <i>Artemisia annua</i> 4-Hydroxy-3-Methylbut-2-enyl Diphosphate Reductase 1 Gene (<i>AaHDR1</i>) Differentially Regulate Artemisinin and Terpenoid Biosynthesis.

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