CDNA encoding tropinone reductase-II from Hyoscyamus niger.

Abstract

Two stereospecific NADPH-dependent reductases, TR-I and TR-11, constitute a branching point in the biosynthesis of tropane alkaloids. TR-I catalyzes the stereospecific reduction of tropinone to tropine (Koelen and Gross, 1982), whereas TR-I1 reduces tropinone to pseudotropine (Drager et al., 1988). We previously characterized TRs that had been purified from cultured roots of Hyoscyamus niger (Hashimoto et al., 1992) and showed that the two TRs had both common and different biochemical and kinetic properties. To obtain a better understanding of the structure and evolutionary relationship of these reductases, we isolated cDNA clones coding for TR-I1 from H. niger (Table I). An intemal amino acid sequence was found in both TR-I and TR-I1 that had been purified from the cultured roots of Datura stramonium and H. niger, respectively (Nakajima et al., 1993). An oligonucleotide probe corresponding to this sequence was synthesized and used to screen the cDNA library from cultured roots of H. niger. DNA sequencing analysis revealed that a11 four of the isolated cDNA clones encoded the TR-I1 polypeptide, probably due to the low concentration of the TR-I transcript in this genus. None of the cDNA clones contained a full ORF; some lacked the amino-terminal part and others the carboxy-terminal part and the 3‘ nontranslated region. Because the nucleotide sequence of the overlapping part (0.4-0.8 kb) matched perfectly, we concluded that these clones were derived from a single gene. The combined nucleotide sequence (1049 bp) contained a 783-bp ORF coding for a polypeptide composed of 260 amino acids, and the calculated mo1 wt of 28,436 agreed well with the molecular mass for the TR-I1 subunit (29 kD) that had been purified from H. niger (Hashimoto et al., 1992). The isolated cDNA was expressed in Escherichia coli as a fusion protein to /3-galactosidase under the control of the Zac promoter. The fusion protein was induced by isopropyl-P-Dthiogalactopyranoside, and the bacterial lysate was assayed for TR activities as described elsewhere (Nakajima et al., 1993). The fusion protein catalyzed the same highly stereospecific reduction of tropinone as the TR-I1 from H. niger; pseudotropine was the sole reaction product detected. The deduced amino acid sequence of TR-I1 from H. niger is highly homologous to that from D. stramonium (Nakajima