Progesterone 5β-reductases (P5βR; EC 1.3.99.6) encoded by Vein Patterning 1 (VEP1) genes are capable of reducing the CC double-bond of a variety of enones enantioselectively. Sequence and activity data of orthologous P5βRs were used to define a set of residues possibly responsible for the large differences in enzyme activity seen between rAtSt5βR and rDlP5βR, recombinant forms of P5βRs from Arabidopsis thaliana and Digitalis lanata, respectively. Tyrosine-156, asparagine-205 and serine-248 were identified as hot spots in the rDlP5βR responsible for its low catalytic efficiency. These positions were individually substituted for amino acids found in the strong rAtSt5βR in the corresponding sites. Kinetic constants were determined for rDlP5βR and its mutants as well as for rAtSt5βR using progesterone and 2-cyclohexen-1-one as substrates. Enzyme mutants in which asparagine-205 was substituted for methionine or alanine showed considerably lower km and higher K(cat)/k(m) values than the wild-type DlP5βR, approaching the catalytic efficiency of strong P5βRs. The introduced mutations not only lead to an improved capability to reduce progesterone but also to altered substrate preference. Our findings provided structural insights into the differences seen among the natural P5βRs with regard to their substrate preferences and catalytic efficiencies.