Abstract

Hydroxymethylbutenyl 4-diphosphate reductase (HDR) catalyzes the last reaction of the methylerythritol phosphate (MEP) pathway for the biosynthesis of artemisinin precursors, a branching step that separately produces isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) in a ratio of 5:1 to 6:1. The full-length cDNA sequence of HDR was cloned and characterized from Artemisia annua L. for the first time. The new cDNA was designated as AaHDR (GenBank accession No.: GQ119345). The full-length cDNA ofAaHDR was 1640 bp containing a 1368 bp open reading frame (ORF) encoding a polypeptide of 455 amino acids with a calculated molecular mass of 51.3 KDa and an isoelectric point of 5.63. Comparative and bioinformatic analysis revealed that AaHDR had extensive homology with HDRs from other plant species and contained a conserved transit peptide for plastids. The phylogenetic analysis indicated that all HDRs could be divided into three groups and AaHDR belonged to plant HDRs family. Then the homology-based structural modeling of AaHDR showed that AaHDR had the typical structure of HDR fromA. aeolicus, which adopted a cloverleaf or trefoil-like structure with each monomer in the dimer containing three alpha/beta domains surrounding a central [Fe₃S₄] cluster ligated to Cys13, Cys96 and Cys193. Finally, AaHDR was transformed into the E. coli HDR mutant strain MG1655 ara< >HDR, which was able to rescue the lethal phenotype of the E. coliHDR mutant strain MG1655 ara-HDR. This confirmed that AaHDR had the typical function of HDR gene. The cloning and characterization of AaHDR will be helpful to understand more about the function of HDR at the level of molecular genetics and unveil the biosynthetic mechanism of artemisinin precursors. The present work also provides a candidate gene for metabolic engineering of the artemisinin biosynthesis pathway in A. annua.

Key words: Artemisia annua L., hydroxymethylbutenyl 4-diphosphate reductase, cloning, characterization, functional complementation.