Alexandra Binter

Entropy-driven binding of opioid peptides induces a large domain motion in human dipeptidyl peptidase III

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 enzymes 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.

Identification of promiscuous ene-reductase activity by mining structural databases using active site constellations

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.

Collapse of the native structure caused by a single amino acid exchange in human NAD(P)H:quinone oxidoreductase1

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.

PLP-dependent enzymes as potential drug targets for protozoan diseases

The chemical properties of the B(6) vitamers are uniquely suited for wide use as cofactors in essential reactions, such as decarboxylations and transaminations. This review addresses current efforts to explore vitamin B(6) dependent enzymatic reactions as drug targets. Several current targets are described that are found amongst these enzymes. The focus is set on diseases caused by protozoan parasites. Comparison across a range of these organisms allows insight into the distribution of potential targets, many of which may be of interest in the development of broad range anti-protozoan drugs. This article is part of a Special Issue entitled: Pyridoxal Phosphate Enzymology.

A single intersubunit salt bridge affects oligomerization and catalytic activity in a bacterial quinone reductase

YhdA, a thermostable NADPH:FMN oxidoreductase from Bacillus subtilis, reduces quinones via a ping‐pong bi‐bi mechanism with a pronounced preference for NADPH. The enzyme occurs as a stable tetramer in solution. The two extended dimer surfaces are packed against each other by a 90° rotation of one dimer with respect to the other. This assembly is stabilized by the formation of four salt bridges between K109 and D137 of the neighbouring protomers. To investigate the importance of the ion pair contacts, the K109L and D137L single replacement variants, as well as the K109L/D137L and K109D/D137K double replacement variants, were generated, expressed, purified, crystallized and biochemically characterized. The K109L and D137L variants form dimers instead of tetramers, whereas the K109L/D137L and K109D/D137K variants appear to exist in a dimer–tetramer equilibrium in solution. The crystal structures of the K109L and D137L variants confirm the dimeric state, with the K109L/D137L and K109D/D137K variants adopting a tetrameric assembly. Interestingly, all protein variants show a drastically reduced quinone reductase activity in steady‐state kinetics. Detailed analysis of the two half reactions revealed that the oxidative half reaction is not affected, whereas reduction of the bound FMN cofactor by NADPH is virtually abolished. Inspection of the crystal structures indicates that the side chain of K109 plays a dual role by forming a salt bridge to D137, as well as stabilizing a glycine‐rich loop in the vicinity of the FMN cofactor. In all protein variants, this glycine‐rich loop exhibits a much higher mobility, compared to the wild‐type. This appears to be incompatible with NADPH binding and thus leads to abrogation of flavin reduction.

The Structure of Glycerol Trinitrate Reductase NerA from Agrobacterium radiobacter Reveals the Molecular Reason for Nitro- and Ene-Reductase Activity in OYE Homologues.

In recent years, Old Yellow Enzymes (OYEs) and their homologues have found broad application in the efficient asymmetric hydrogenation of activated CC bonds with high selectivities and yields. Members of this class of enzymes have been found in many different organisms and are rather diverse on the sequence level, with pairwise identities as low as 20 %, but they exhibit significant structural similarities with the adoption of a conserved (αβ)8‐barrel fold. Some OYEs have been shown not only to reduce CC double bonds, but also to be capable of reducing nitro groups in both saturated and unsaturated substrates. In order to understand this dual activity we determined and analyzed X‐ray crystal structures of NerA from Agrobacterium radiobacter, both in its apo form and in complex with 4‐hydroxybenzaldehyde and with 1‐nitro‐2‐phenylpropene. These structures, together with spectroscopic studies of substrate binding to several OYEs, indicate that nitro‐containing substrates can bind to OYEs in different binding modes, one of which leads to CC double bond reduction and the other to nitro group reduction.

Characterization of the PLP-dependent aminotransferase NikK from Streptomyces tendae and its putative role in nikkomycin biosynthesis

As inhibitors of chitin synthase, nikkomycins have attracted interest as potential antibiotics. The biosynthetic pathway to these peptide nucleosides in Streptomyces tendae is only partially known. In order to elucidate the last step of the biosynthesis of the aminohexuronic building block, we have heterologously expressed a predicted aminotransferase encoded by the gene nikK from S. tendae in Escherichia coli. The purified protein, which is essential for nikkomycin biosynthesis, has a pyridoxal‐5′‐phosphate cofactor bound as a Schiff base to lysine 221. The enzyme possesses aminotransferase activity and uses several standard amino acids as amino group donors with a preference for glutamate (Glu > Phe > Trp > Ala > His > Met > Leu). Therefore, we propose that NikK catalyses the introduction of the amino group into the ketohexuronic acid precursor of nikkomycins. At neutral pH, the UV‐visible absorbance spectrum of NikK has two absorbance maxima at 357 and 425 nm indicative of the presence of the deprotonated and protonated aldimine with an estimated pKa of 8.3. The rate of donor substrate deamination is faster at higher pH, indicating that an alkaline environment favours the deamination reaction.

Catalytic competence, structure and stability of the cancer‐associated R139W variant of the human NAD(P)H:quinone oxidoreductase 1 (NQO1)

The human NAD(P)H:quinone oxidoreductase 1 (NQO1; EC1.6.99.2) is an essential enzyme in the antioxidant defence system. Furthermore, NQO1 protects tumour suppressors like p53, p33ING1b and p73 from proteasomal degradation. The activity of NQO1 is also exploited in chemotherapy for the activation of quinone‐based treatments. Various single nucleotide polymorphisms are known, such as NQO1*2 and NQO1*3 yielding protein variants of NQO1 with single amino acid replacements, i.e. P187S and R139W, respectively. While the former NOQ1 variant is linked to a higher risk for specific kinds of cancer, the role, if any, of the arginine 139 to tryptophan exchange in disease development remains obscure. On the other hand, mitomycin C‐resistant human colon cancer cells were shown to harbour the NQO1*3 variant resulting in substantially reduced enzymatic activity. However, the molecular cause for this decrease remains unclear. In order to resolve this issue, recombinant NQO1 R139W has been characterized biochemically and structurally. In this report, we show by X‐ray crystallography and 2D‐NMR spectroscopy that this variant adopts the same structure both in the crystal as well as in solution. Furthermore, the kinetic parameters obtained for the variant are similar to those reported for the wild‐type protein. Similarly, thermostability of the variant was only slightly affected by the amino acid replacement. Therefore, we conclude that the previously reported effects in human cancer cells cannot be attributed to protein stability or enzyme activity. Instead, it appears that loss of exon 4 during maturation of a large fraction of pre‐mRNA is the major reason of the observed lack of enzyme activity and hence reduced activation of quinone‐based chemotherapeutics.

Structural and Functional Characterization of NikO, an Enolpyruvyl Transferase Essential in Nikkomycin Biosynthesis

Background: Nikkomycins are potent antibiotics. NikO is a key enzyme in their biosynthesis. Results: NikO is structurally closely related to other enolpyruvyl transferases and is inhibited by fosfomycin. Conclusion: Conservation of important active site residues indicates mechanistic similarities to MurA. Significance: The structure of NikO is the first of an enzyme participating in the formation of the aminohexuronic acid moiety in nikkomycin biosynthesis. Nikkomycins are peptide-nucleoside compounds with fungicidal, acaricidal, and insecticidal properties because of their strong inhibition of chitin synthase. Thus, they are potential antibiotics especially for the treatment of immunosuppressed patients, for those undergoing chemotherapy, or after organ transplants. Although their chemical structure has been known for more than 30 years, only little is known about their complex biosynthesis. The genes encoding for proteins involved in the biosynthesis of the nucleoside moiety of nikkomycins are co-transcribed in the same operon, comprising the genes nikIJKLMNO. The gene product NikO was shown to belong to the family of enolpyruvyl transferases and to catalyze the transfer of an enolpyruvyl moiety from phosphoenolpyruvate to the 3′-hydroxyl group of UMP. Here, we report activity and inhibition studies of the wild-type enzyme and the variants C130A and D342A. The x-ray crystal structure revealed differences between NikO and its homologs. Furthermore, our studies led to conclusions concerning substrate binding and preference as well as to conclusions about inhibition/alkylation by the antibiotic fosfomycin.

Asymmetric Reductive Carbocyclization Using Engineered Ene Reductases

Abstract 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.