Opioid peptides are involved in various essential physiological processes, most notably nociception. Dipeptidyl peptidase III (DPP III) is one of the most important enkephalin-degrading enzymes associated with the mammalian pain modulatory system. Here we describe the X-ray structures of human DPP III and its complex with the opioid peptide tynorphin, which rationalize the enzyme's substrate specificity and reveal an exceptionally large domain motion upon ligand binding. Microcalorimetric analyses point at an entropy-dominated process, with the release of water molecules from the binding cleft ("entropy reservoir") as the major thermodynamic driving force. Our results provide the basis for the design of specific inhibitors that enable the elucidation of the exact role of DPP III and the exploration of its potential as a target of pain intervention strategies.isothermal titration calorimetry | metallopeptidase | peptide binding | X-ray crystallography T he endogenous opioid system, composed of opioid peptides and their receptors, modulates a large number of physiological processes, such as endocrine and immune function, gastrointestinal motility, respiration, reward, stress, complex social behavior (e.g., sexual activity), vulnerability to drug addiction, and most notably the procession and transmission of pain stimuli (nociception) (1, 2). Two major types of endogenous opioid peptides are those containing enkephalin sequences at the N terminus (TyrGly-Gly-Phe-Met/Leu) (3) and, more recently identified, endomorphins 1 and 2 (Tyr-Pro-Trp/Phe-Phe-NH 2 ) (4, 5). Knowledge and control over synthesis and degradation pathways of this class of molecules is prerequisite for the development of new therapies that target pertinent physiological processes.Dipeptidyl peptidase III (DPP III), also known as enkephalinase B, is an enkephalin-degrading enzyme that cleaves dipeptides sequentially from the N termini of substrates (6). All DPP IIIs described thus far contain the unique zinc-binding motif HEXXGH characteristic of metallopeptidase family M49 (7). Enzymes from several human and animal tissues, as well as from lower eukaryotes, were purified and biochemically characterized (8, 9). DPP III is largely found as a cytosolic protein, although membrane association in rat brain and Drosophila melanogaster has been described (10, 11). The 3D structure of the yeast ortholog has recently been determined, revealing a unique protein fold with two lobes forming a wide-open substrate-binding cleft (12). The lack of structural information on peptide complexes, however, left the question of substrate specificity largely unanswered.DPP III purified from monkey brain microsomes is strongly inhibited by the neuropeptide spinorphin (Leu-Val-Val-Tyr-ProTrp-Thr), an endogenous factor isolated from bovine spinal cord that also inhibits other enkephalin-degrading enzymes, such as neutral endopeptidase (NEP, neprilysin), aminopeptidase, and angiotensin-converting enzyme (13). Because of a different mode of action compared with morphine, spinorp...

Human NAD(P)H:quinone oxidoreductase 1 (NQO1) is essential for the antioxidant defense system, stabilization of tumor suppressors (e.g. p53, p33, and p73), and activation of quinone-based chemotherapeutics. Overexpression of NQO1 in many solid tumors, coupled with its ability to convert quinone-based chemotherapeutics into potent cytotoxic compounds, have made it a very attractive target for anticancer drugs. A naturally occurring single-nucleotide polymorphism (C609T) leading to an amino acid exchange (P187S) has been implicated in the development of various cancers and poor survival rates following anthracyclin-based adjuvant chemotherapy. Despite its importance for cancer prediction and therapy, the exact molecular basis for the loss of function in NQO1 P187S is currently unknown. Therefore, we solved the crystal structure of NQO1 P187S. Surprisingly, this structure is almost identical to NQO1. Employing a combination of NMR spectroscopy and limited proteolysis experiments, we demonstrated that the single amino acid exchange destabilized interactions between the core and C-terminus, leading to depopulation of the native structure in solution. This collapse of the native structure diminished cofactor affinity and led to a less competent FAD-binding pocket, thus severely compromising the catalytic capacity of the variant protein. Hence, our findings provide a rationale for the loss of function in NQO1 P187S with a frequently occurring single-nucleotide polymorphism.

The exploitation of catalytic promiscuity and the application of de novo design have recently opened the access to novel, non-natural enzymatic activities. Here we describe a structural bioinformatic method for predicting catalytic activities of enzymes based on three-dimensional constellations of functional groups in active sites (‘catalophores’). As a proof-of-concept we identify two enzymes with predicted promiscuous ene-reductase activity (reduction of activated C–C double bonds) and compare them with known ene-reductases, that is, members of the Old Yellow Enzyme family. Despite completely different amino acid sequences, overall structures and protein folds, high-resolution crystal structures reveal equivalent binding modes of typical Old Yellow Enzyme substrates and ligands. Biochemical and biocatalytic data show that the two enzymes indeed possess ene-reductase activity and reveal an inverted stereopreference compared with Old Yellow Enzymes for some substrates. This method could thus be a tool for the identification of viable starting points for the development and engineering of novel biocatalysts.

Ene reductases from the Old Yellow Enzyme (OYE) family reduce the C=C double bond in α,β‐unsaturated compounds bearing an electron‐withdrawing group, for example, a carbonyl group. This asymmetric reduction has been exploited for biocatalysis. Going beyond its canonical function, we show that members of this enzyme family can also catalyze the formation of C−C bonds. α,β‐Unsaturated aldehydes and ketones containing an additional electrophilic group undergo reductive cyclization. Mechanistically, the two‐electron‐reduced enzyme cofactor FMN delivers a hydride to generate an enolate intermediate, which reacts with the internal electrophile. Single‐site replacement of a crucial Tyr residue with a non‐protic Phe or Trp favored the cyclization over the natural reduction reaction. The new transformation enabled the enantioselective synthesis of chiral cyclopropanes in up to >99 % ee.