Truncation of N-terminal regions of Digitalis lanata progesterone 5β-reductase alters catalytic efficiency and substrate preference.

N-Terminal truncated forms of progesterone 5β-reductase (P5βR) were synthesized taking a full-length cDNA encoding for Digitalis lanata P5βR with a hexa-histidine tag attached at the C-terminus (rDlP5βRc) as the starting point. Four pETite-c-His/DlP5βR constructs coding for P5βR derivatives truncated in the N-terminal region, termed rDlP5βRcn-10, rDlP5βRcn-20, rDlP5βRcn-30, and rDlP5βRcn-40 were obtained by site-directed mutagenesis. The cDNAs coding for full-length rDlP5βRc, rDlP5βRcn-10 and rDlP5βRcn-20 were over-expressed in Escherichia coli and the respective enzymes were soluble and catalytically active (progesterone and 2-cyclohexen-1-one as substrates). GST-tagged recombinant DlP5βR (rDlP5βR-GST) and rDlP5βR-GSTr, with the GST-tag removed by protease treatment were produced as well and served as controls. The Km values and substrate preferences considerably differed between the various DlP5βR derivatives. As for the C-terminal His-tagged rDlP5βR the catalytic efficiency for progesterone was highest for the full-length rDlP5βRc whereas the N-terminal truncated forms preferred 2-cyclohexen-1-one as the substrate. Affinity tags and artifacts resulting from the cloning strategy used may alter substrate specificity. Therefore enzyme properties determined with recombinant proteins should not be used to infer in vivo scenarios and should be considered for each particular case.