Opposite stereospecificity of two tropinone reductases is conferred by the substrate-binding sites.

Abstract

Two tropinone reductases (TRs) catalyze opposite stereospecific reductions at a branching point in the biosynthetic pathway of tropane alkaloids. The two TRs, TR-I and TR-II, reduce the 3-keto group of the common substrate tropinone stereospecifically to 3 alpha- and 3 beta-hydroxy groups, to produce the stereoisomeric alkamines tropine and pseudotropine, respectively. Sixteen chimeric TR enzymes were expressed in Escherichia coli, and their stereospecificities, substrate specificities, and Km values for tropinone were compared with those of the wild-type enzymes. Stereospecificity and substrate specificity of the chimeric enzymes were closely correlated, and the carboxyl-terminal peptides of about 120 amino acid residues, in which 53 residues were different between TR-I and TR-II, were shown to determine both specificities. Further dissection of these peptide segments resulted in either enzymes with both TR activities or inactive enzymes. The substrate binding affinity of many chimeric enzymes was much lower than that of wild-type enzymes. These results indicate that the stereospecificity of TR is determined by the orientation of tropinone at the substrate-binding site, which is composed mainly of the carboxyl-terminal half region, and also that the amino-terminal half region constitutes the NADPH-binding site as postulated for short chain nonmetal dehydrogenases.