Abstract
Progesterone 5beta-reductase (5beta-POR) catalyzes the stereospecific reduction of progesterone to 5beta-pregnane-3,20-dione and is a key enzyme in the biosynthetic pathway of cardenolides in Digitalis (foxglove) plants. Sequence considerations suggested that 5beta-POR is a member of the short chain dehydrogenase/reductase (SDR) family of proteins but at the same time revealed that the sequence motifs that in standard SDRs contain the catalytically important residues are missing. Here we present crystal structures of 5beta-POR from Digitalis lanata in complex with NADP(+) at 2.3A and without cofactor bound at 2.4A resolution together with a model of a ternary complex consisting of 5beta-POR, NADP(+), and progesterone. Indeed, 5beta-POR displays the fold of an extended SDR. The architecture of the active site is, however, unprecedented because none of the standard catalytic residues are structurally conserved. A tyrosine (Tyr-179) and a lysine residue (Lys-147) are present in the active site, but they are displayed from novel positions and are part of novel sequence motifs. Mutating Tyr-179 to either alanine or phenylalanine completely abolishes the enzymatic activity. We propose that the distinct topology reflects the fact that 5beta-POR reduces a conjugated double bond in a steroid substrate via a 1-4 addition mechanism and that this requires a repositioning of the catalytically important residues. Our observation that the sequence motifs that line the active site are conserved in a number of bacterial and plant enzymes of yet unknown function leads us to the proposition that 5beta-POR defines a novel class of SDRs.
Highlights
The beneficial effects of cardenolides, known as cardiac glycosides or cardiotonic steroids, are well documented, and they have been applied for the treatment of cardiac insufficiencies for centuries [1,2,3]
The question arises whether 5-POR defines a novel class of short chain dehydrogenase/reductase (SDR) with a different set of sequence motifs and conserved residues in the catalytic site and that might be characterized by a distinct reaction mechanism
The Structure of 5-POR—The structure of 5-POR from D. lanata was solved in complex with the cofactor NADPϩ at a resolution of 2.3 Å (Rfactor ϭ 17.0%, Rfree ϭ 21.3%) and with no cofactor bound at 2.4 Å resolution (Rfactor ϭ 20.2%, Rfree ϭ 24.9%) (Table 1)
Summary
Protein Production and Purification—To produce recombinant 5-POR from D. lanata, we slightly modified previously published protocols [5, 23]. Molecular Modeling of the Ternary Protein Cofactor Substrate Complex—Because any attempts to produce crystals of 5-POR in complex with either progesterone, cortisol, or 4-androstene-3,17-dione remained unsuccessful regardless whether the cofactor NADPϩ was present or not, we computationally docked the substrate progesterone into the binding site. For this purpose, the protein design algorithms of the inhouse program MUMBO were supplemented with a flexible ligand-handling routine to identify the energetically most favorable interaction between progesterone and the protein [32]. A more detailed description of the method and validation calculations are provided in supplemental Figs. 1 and 2 and supplemental Tables I and II)
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