Molecular cloning and characterization of a tropinone reductase from Dendrobium nobile Lindl

Abstract

A cDNA sequence that encodes a peptide with similarity to known tropinone reductases (TR) was cloned from Dendrobium nobile Lindl. The full coding region of the gene (DnTR1) is 804bp in length which encodes a putative peptide consisting of 268 amino acids. Phylogenetic analysis showed that DnTR1 was a novel member of the TR family and evolutionarily distant from those well-characterized subgroups of TRs, suggesting that DnTR1 may have distinct characteristics. Structural modeling found that DnTR1 had a similar electrostatic environment at the inner molecular surface of the substrate binding pocket with TRI encoded by Datura stramonium (DsTRI). Catalytic activity assay with recombinant protein demonstrated that DnTR1 was able to reduce tropinone, 3-quinuclidinone hydrochloride, and 4-methylcyclohexanone using NADPH as coenzyme. Gene expression profiling by qRT-PCR revealed that the DnTR1 transcript was expressed in all three vegetative organs (leaves, stems and roots) of D. nobile with the highest expression level in roots. The expression of DnTR1 mRNA was enhanced 9.5 times (P<0.01) by treatment of methyl jasmonate at 24h, but not affected by salicylic acid and sodium nitroprusside treatments, indicating that DnTR1 regulation may be involved in a jasmonate-dependent pathway.