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

摘要: 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 3-keto group common substrate stereospecifically to 3 alpha- beta-hydroxy groups, produce stereoisomeric alkamines tropine pseudotropine, respectively. Sixteen chimeric TR enzymes were expressed Escherichia coli, their stereospecificities, specificities, Km values for compared with those wild-type enzymes. Stereospecificity specificity closely correlated, carboxyl-terminal peptides about 120 amino acid residues, which 53 residues different between shown determine both specificities. Further dissection these peptide segments resulted either activities or inactive binding affinity many was much lower than that These results indicate stereospecificity is determined by orientation substrate-binding site, composed mainly half region, also amino-terminal region constitutes NADPH-binding site as postulated short chain nonmetal dehydrogenases.