Molecular cloning, expression and characterization of recombinant 1-deoxy- d-xylulose-5-phosphate reductoisomerase from Coleus forskohlii Briq.

Abstract

1-deoxy- d-xylulose-5-phosphate reductoisomerase (DXR) catalyses the first committed step in the production of isopentenyl diphosphate (IPP) according to the recently identified plastidial 2 C-methyl- d-erythritol 4-phosphate (MEP) pathway. Forskolin, the major active diterpenoid component in Coleus forskohlii is synthesised via the MEP pathway. Herein, we present the investigation of a cDNA encoding C. forskohlii DXR, its heterologous expression in Escherichia coli and an examination of its properties in vitro. The full-length cDNA sequence of C. forskohlii DXR contains an open reading frame (ORF) of 1407 nucleotides encoding a peptide of 469 amino acids, resulting in an estimated molecular mass of 50.8 kDa. The K m and V max of C. forskohlii DXR were determined to be 147.2 μM and 0.3 units mg protein −1, respectively. Fosmidomycin, a specific inhibitor of DXR, showed dose-dependent inhibition with an IC 50 of 0.45 μM. A gene expression study using a C. forskohlii plant culture showed that DXR is strongly expressed in leaves, less in stems and at the limit of detectability in roots. The presence of fosmidomycin in C. forskohlii plant culture resulted in a decrease in forskolin production in roots. Therefore, we conclude that if C. forskohlii DXR is involved in the biosynthesis of forskolin, it is primarily synthesised in the leaves, and later accumulates in stems and roots.