Functional characterization of a novel tropinone reductase-like gene in Dendrobium nobile Lindl

Dendrobium nobile, a herbal medicine plant, contains many important alkaloids and other secondary metabolites with pharmacological and clinical effects. However, the biosynthetic pathway of these secondary metabolites is largely unknown. In present study, a cDNA sequence (DnTR2) that encodes a peptide with high similarity to known tropinone reductase (TR) was cloned from D. nobile Lindl. Sequence comparison and phylogenetic analysis showed that DnTR2 was evolutionarily distant from those well-characterized subgroups of TRs. qRT-PCR revealed that DnTR2 was expressed constitutively in all three vegetative organs (leaves, stems and roots) and was regulated by methyl jasmonate (MeJA), salicylic acid (SA) and nitrogen oxide (NO). Catalytic activity analysis using recombinant protein found that DnTR2 was not able to reduce tropinone, but reduced the two structural analogs of tropinone, 3-quinuclidinone hydrochloride and 4-methylcyclohexanone. Structural modeling and comparison suggested that the substrate specificity of TRs may not be determined by their phylogenetic relationships but by the amino acids that compose the substrate binding pocket. To verify this hypothesis, a site-directed mutagenesis was performed and it successfully restored the DnTR2 with tropinone reduction activity. Our results also showed that the substrate specificity of TRs was determined by a few residues that compose the substrate binding pocket which may have an important role for directed selecting of TRs with designated substrate specificities.