2 TROPINONE REDUCTASES WITH DIFFERENT STEREOSPECIFICITIES ARE SHORT-CHAIN DEHYDROGENASES EVOLVED FROM A COMMON ANCESTOR

In the biosynthetic pathway of tropane alkaloids, tropinone reductase (EC 1.1.1.236) (TR)-I and TR-II, respectively, reduce a common substra te, tropinone, stereospecifically to the stereoisomeric alkamines trop ine and pseudotropine (psi-tropine). cDNA clones coding for TR-I and T R-II, as well as a structurally related cDNA clone with an unknown fun ction, were isolated from the solanaceous plant Datura stramonium. The cDNA clones for TR-I and TR-II encode polypeptides containing 273 and 260 amino acids, respectively, and when these clones were expressed i n Escherichia coli, the recombinant TRs showed the same strict stereos pecificity as that observed for the native TRs that had been isolated from plants. The deduced amino acid sequences of the two clones showed an overall identity of 64% in 260-amino acid residues and also shared significant similarities with enzymes in the short-chain, nonmetal de hydrogenase family. Genomic DNA-blot analysis detected the TR-encoding genes in three tropane alkaloid-producing solanaceous species but did not detect them in tobacco. We discuss how the two TRs may have evolv ed to catalyze the opposite stereospecific reductions.