Reversible, ß-sheet-dependent self-assembly of the phosphoprotein phosvitin is controlled by the concentration and valency of cations.

The hyperphosphorylated protein phosvitin (PV) undergoes a pH-dependent transition between PII- and ß-sheet secondary structures, a process deemed crucial for its role in the promotion of biogenic apatite formation. The transition occurs surprisingly slowly (minutes to hours). This is consistent with a slow aggregation process involving ionic interactions of charged groups on the protein surface. Herein, we determined the associated transition pK values and time constants through matrix least-squares (MLS) global fitting of a series of pH- and time-dependent circular dichroism (CD) spectra recorded in the presence of different mono-, bi- and trivalent cations. Supporting our results with dynamic light scattering data, we clearly identified a close correlation of ß-sheet transition and the formation of small aggregates at low pH. This process is inhibited in the presence of all tested cations with the strongest effects for trivalent cations (Fe3+ and Al3+). In the presence of Ca2+ and Mg2+, larger higher-order particles are formed from PV in the ß-sheet conformation, as identified from the interpretation of differential scattering observed in the CD spectra. Our observations are consistent with the existence of a multi-step equilibrium between aggregated and non-aggregated species of PV. The equilibrium is highly sensitive to the environment pH and salt concentration with exceptional behavior in the presence of divalent cations such as Ca2+ and Mg2+.

Dynamic Structural Changes and Thermodynamics in Phase Separation Processes of an Intrinsically Disordered-Ordered Protein Model.

Elastin-like proteins (ELPs) are biologically important proteins and models for intrinsically disordered proteins (IDPs) and dynamic structural transitions associated with coacervates and liquid-liquid phase transitions. However, the conformational status below and above coacervation temperature and its role in the phase separation process is still elusive. Employing matrix least-squares global Boltzmann fitting of the circular dichroism spectra of the ELPs (VPGVG)20 , (VPGVG)40 , and (VPGVG)60 , we found that coacervation occurs sharply when a certain number of repeat units has acquired ß-turn conformation (in our sequence setting a threshold of approx. 20 repeat units). The character of the differential scattering of the coacervate suspensions indicated that this fraction of ß-turn structure is still retained after polypeptide assembly. Such conformational thresholds may also have a role in other protein assembly processes with implications for the design of protein-based smart materials.

Enzymatic Asymmetric Reduction of Unfunctionalized C=C Bonds with Archaeal Geranylgeranyl Reductases.

The asymmetric reduction of activated C=C bonds such as enones is well established for non-enzymatic methods as well as in biocatalysis. However, the asymmetric reduction of unfunctionalized C=C bonds is mainly performed with transition metal catalysts whereas biocatalytic approaches are lacking. We have tested two FAD-dependent archaeal geranylgeranyl reductases (GGR) for the asymmetric reduction of isolated C=C bonds. The reduction of up to four double bonds in terpene chains with different chain lengths and head groups was confirmed. Methyl-branched E-alkenes were chemoselectively reduced in the presence of cyclic, terminal or activated alkenes. Using a removable succinate "spacer", farnesol and geraniol could be quantitatively reduced (>99 %). The reduction is strictly (R)-selective (enantiomeric excess >99 %). Hence, GGRs are promising biocatalysts for the asymmetric reduction of unactivated isolated C=C bonds, opening new opportunities for the synthesis of enantiopure branched alkyl chains.

Targeting HSP90 dimerization via the C terminus is effective in imatinib-resistant CML and lacks the heat shock response.

Heat shock protein 90 (HSP90) stabilizes many client proteins, including the BCR-ABL1 oncoprotein. BCR-ABL1 is the hallmark of chronic myeloid leukemia (CML) in which treatment-free remission (TFR) is limited, with clinical and economic consequences. Thus, there is an urgent need for novel therapeutics that synergize with current treatment approaches. Several inhibitors targeting the N-terminal domain of HSP90 are under investigation, but side effects such as induction of the heat shock response (HSR) and toxicity have so far precluded their US Food and Drug Administration approval. We have developed a novel inhibitor (aminoxyrone [AX]) of HSP90 function by targeting HSP90 dimerization via the C-terminal domain. This was achieved by structure-based molecular design, chemical synthesis, and functional preclinical in vitro and in vivo validation using CML cell lines and patient-derived CML cells. AX is a promising potential candidate that induces apoptosis in the leukemic stem cell fraction (CD34+CD38-) as well as the leukemic bulk (CD34+CD38+) of primary CML and in tyrosine kinase inhibitor (TKI)-resistant cells. Furthermore, BCR-ABL1 oncoprotein and related pro-oncogenic cellular responses are downregulated, and targeting the HSP90 C terminus by AX does not induce the HSR in vitro and in vivo. We also probed the potential of AX in other therapy-refractory leukemias. Therefore, AX is the first peptidomimetic C-terminal HSP90 inhibitor with the potential to increase TFR in TKI-sensitive and refractory CML patients and also offers a novel therapeutic option for patients with other types of therapy-refractory leukemia because of its low toxicity profile and lack of HSR.

Hidden Specificities in Enzyme Catalysis: Structural Basis of Substrate Structure-Selectivity Relationship of a Ketoreductase.

Enzymes often convert both physiological and non-physiological substrates with high stereoselectivity; yet, for some enzymes, opposite product chirality is observed. A possible explanation is the existence of hidden specificities becoming apparent when non-physiological substrates confer different substrate-enzyme interactions than the physiological substrate. To test this hypothesis, a series of α-methylated ß-keto esters were converted with Tyl-KR1, a ketoreductase from polyketide synthesis in Streptomyces fradiae. The conversions of six substrates with different physicochemical properties exhibited enantioselectivities ranging from 84 % ee for R,R to 84 % ee for S,S, yet high and uniform diastereoselectivity (anti, d.r.>9:1). The exchange of a single atom, namely an oxygen ester instead of a thioester, led to almost complete loss of enantioselectivity (<5 % ee). An additional S,S-selective binding mode as a hidden specificity in Tyl-KR1 has been identified through molecular modeling and site-directed mutagenesis.

Studying NAD(P)H cofactor-binding to alcohol dehydrogenases through global analysis of circular dichroism spectra.

The initial step in reactions catalyzed by NAD(P)H-dependent alcohol dehydrogenases (ADHs) is the binding of the cofactor to the active site. To study this process, we measured NAD(P)H concentration-dependent circular dichroism (CD) in the presence of purified enzymes (ADH from horse liver, HLADH; ADH-A from Rhodococcus ruber; YGL157w from Saccharomyces cerevisiae) or enzyme-containing whole cell extract (ADH from Lactobacillus brevis, LbADH). We determined the proportions of binding and non-binding NAD(P)H and the associated dissociation constants (Kd) from matrix least-squares global fitting of law of mass action-derived model. Furthermore, the fitting allowed the back calculation of CD spectra corresponding to the cofactor in its bound conformation. With increasing pH and/or increasing ionic strength, we detected an increase in Kd for the NADH·HLADH complex with the shape of the bound cofactor conformation spectrum remaining unaffected. While the bound cofactor spectrum for the ADH-A·NADH complex was similar to that for HLADH, the corresponding spectra obtained for the NADPH-dependent enzymes YGL157w and LbADH exhibited opposite signs of the most prominent band. In comparison to CD spectra calculated on cofactor geometries from the crystal structures at the sTD-DFT level, we found that the sign of the bound cofactor spectrum correlates with the orientation of the nicotinamide ring of the cofactor in the active site. These results demonstrate the usefulness of the global analysis of cofactor titration CD spectra to study the role of cofactor binding and its geometry in ADH catalysis.

Xanthocidin Derivatives from the Endophytic Streptomyces sp. AcE210 Provide Insight into Xanthocidin Biosynthesis.

Xanthocidin and six new derivatives were isolated from the endophytic Streptomyces sp. AcE210. Their planar structures were elucidated by 1D and 2D NMR spectroscopy as well as by HRMS. The absolute configuration of one compound was determined by using vibrational circular dichroism spectroscopy (VCD). The structural similarities of xanthocidin and some of the isolated xanthocidin congeners to the methylenomycins A, B, and C suggested that the biosynthesis of these compounds might follow a similar route. Feeding studies with isotopically labelled [13 C5 ]-l-valine showed that instead of utilizing acetyl-CoA as starter unit, which has been proposed for the methylenomycin biosynthesis, Streptomyces sp. AcE210 employs an isobutyryl-CoA starter unit, resulting in a branched side chain in xanthocidin. Further evidence for a comparable biosynthesis was given by the analysis of the genome sequence of Streptomyces sp. AcE210 that revealed a cluster of homologues to the mmy genes involved in methylenomycin biosynthesis.

Enantioselective Enzymatic Naphthoyl Ring Reduction.

Birch reductions of aromatic hydrocarbons by means of single-electron-transfer steps depend on alkali metals, ammonia, and cryogenic reaction conditions. In contrast, 2-naphthoyl-coenzyme A (2-NCoA) and 5,6-dihydro-2-NCoA (5,6-DHNCoA) reductases catalyze two two-electron reductions of the naphthoyl-ring system to tetrahydronaphthoyl-CoA at ambient temperature. Using a number of substrate analogues, we provide evidence for a Meisenheimer complex-analogous intermediate during 2-NCoA reduction, whereas the subsequent reduction of 5,6-dihydro-2-NCoA is suggested to proceed via an unprecedented cationic transition state. Using vibrational circular dichroism (VCD) spectroscopy, we demonstrate that both enzymatic reductions are highly stereoselective in D2 O, providing an enantioselective pathway to products inaccessible by Birch reduction. Moreover, we demonstrate the power of VCD spectroscopy to determine the absolute configuration of isotopically engendered alicyclic stereocenters.

Broad-Range Spectral Analysis for Chiral Metal Coordination Compounds: (Chiro)optical Superspectrum of Cobalt(II) Complexes.

Chiroptical broad-range spectral analysis extending from UV to mid-IR was employed to study a family of Co(II) N-(1-(aryl)ethyl)salicylaldiminato Schiff base complexes with pseudotetrahedral geometry associated with chirality-at-metal of the Δ/Λ type. While common chiral organic compounds have well-separated absorption and circular dichroism spectra (CD) in the UV/vis and IR regions, chiral Co(II) complexes feature an almost unique continuum of absorption and CD bands, which cover in sequence the UV, visible, near-IR (NIR), and IR regions of the electromagnetic spectrum. They can be collected in a single (chiro)optical superspectrum ranging from the UV (230 nm, 5.4 eV) to the mid-IR (1000 cm-1, 0.12 eV), which offers a fingerprint of the structure and stereochemistry of the metal complexes. Each region of the superspectrum contributes to one piece of information: the NIR-CD region, in combination with TDDFT calculations, allows a reliable assignment of the metal-centered chirality; the UV-CD region facilitates the analysis of the Δ/Λ diastereomeric equilibrium in solution; and the IR-VCD region contains a combination of low-lying metal-centered electronic states (LLES) and ligand-centered vibrations and displays characteristically enhanced and monosignate VCD bands. Circular dichroism in the NIR and IR regions is crucial to reveal the presence of d-d transitions of the Co(II) core which, due to the electric-dipole forbidden character, would be otherwise overlooked in the corresponding absorption spectra.

Extended Catalytic Scope of a Well-Known Enzyme: Asymmetric Reduction of Iminium Substrates by Glucose Dehydrogenase.

NADP(H)-dependent imine reductases (IREDs) are of interest in biocatalytic research due to their ability to generate chiral amines from imine/iminium substrates. In reaction protocols involving IREDs, glucose dehydrogenase (GDH) is generally used to regenerate the expensive cofactor NADPH by oxidation of d-glucose to gluconolactone. We have characterized different IREDs with regard to reduction of a set of bicyclic iminium compounds and have utilized 1 H NMR and GC analyses to determine degree of substrate conversion and product enantiomeric excess (ee). All IREDs reduced the tested iminium compounds to the corresponding chiral amines. Blank experiments without IREDs also showed substrate conversion, however, thus suggesting an iminium reductase activity of GDH. This unexpected observation was confirmed by additional experiments with GDHs of different origin. The reduction of C=N bonds with good levels of conversion (>50 %) and excellent enantioselectivity (up to >99 % ee) by GDH represents a promiscuous catalytic activity of this enzyme.

Addition of a polyhistidine tag alters the regioselectivity of carbonyl reductase S1 from Candida magnoliae.

Studying enzymatic reductions of substrates with more than a single keto group is challenging, as the carbonyl reduction can create a vast array of regio- and stereoisomers. If used as reference compounds, regio- and stereopure hydroxy ketides could facilitate the characterization of reductases with unclear regio- and stereoselectivity. We have combined nonenzymatic and enzymatic reduction and oxidation steps to obtain all four regio- and stereoisomers of tert-butyl hydroxyoxohexanoates in high optical purity (enantiomeric ratio (er) of 99 : 1 for the δ-hydroxy-ß-keto isomers; er of >97 : 3 for the ß-hydroxy-δ-keto isomers). Furthermore, we have prepared seven of the eight possible regioisomers and diastereomers of γ-methylated hydroxyoxohexanoates. These 11 compounds allowed unraveling the complex stereoselectivity of ß,δ-diketo ester reductions catalyzed by carbonyl reductase S1 from Candida magnoliae (CMCR-S1). Our analysis shows that the regio- and stereoselectivity of CMCR-S1-catalyzed reductions is highly sensitive toward modifications at the C-terminus of CMCR-S1: in addition to the expected δ-hydroxy product, the variant with a C-terminal His-tag also led to formation of ß-hydroxy by-products with high optical purity.

Unravelling a Direct Role for Polysaccharide ß-Strands in the Higher Order Structure of Physical Hydrogels.

The mechanical properties of agarose-derived hydrogels depend on the scaffolding of the polysaccharide network. To identify and quantify such higher order structure, we applied Raman optical activity (ROA)-a spectroscopic technique that is highly sensitive toward carbohydrates-on native agarose and chemically modified agarose in the gel phase for the first time. By spectral global fitting, we isolated features that change as a function of backbone carboxylation (28, 40, 50, 60, 80, and 93 %) from other features that remain unchanged. We assigned these spectral features by comparison to ROA spectra calculated for different oligomer models. We found a 60:40 ratio of double- and single-stranded α-helix in the highly rigid hydrogel of native agarose, while the considerably softer hydrogels made from carboxylated agarose use a scaffold of unpaired ß-strands.

Substrate-Determined Diastereoselectivity in an Enzymatic Carboligation.

Thiamine diphosphate-dependent enzymes catalyze the formation of C-C bonds, thereby generating chiral secondary or tertiary alcohols. By the use of vibrational circular dichroism (VCD) spectroscopy we studied the stereoselectivity of carboligations catalyzed by YerE, a carbohydrate-modifying enzyme from Yersinia pseudotuberculosis. Conversion of the non-physiological substrate (R)-3-methylcyclohexanone led to an R,R-configured tertiary alcohol (diastereomeric ratio (dr) >99:1), whereas the corresponding reaction with the S enantiomer gave the S,S-configured product (dr>99:1). This suggests that YerE-catalyzed carboligations can undergo either an R- or an S-specific pathway. We show that, in this case, the high stereoselectivity of the YerE-catalyzed reaction depends on the substrate's preference to acquire a low-energy conformation.

α-Aminoxy Oligopeptides: Synthesis, Secondary Structure, and Cytotoxicity of a New Class of Anticancer Foldamers.

α-Aminoxy peptides are peptidomimetic foldamers with high proteolytic and conformational stability. To gain an improved synthetic access to α-aminoxy oligopeptides we used a straightforward combination of solution- and solid-phase-supported methods and obtained oligomers that showed a remarkable anticancer activity against a panel of cancer cell lines. We solved the first X-ray crystal structure of an α-aminoxy peptide with multiple turns around the helical axis. The crystal structure revealed a right-handed 28 -helical conformation with precisely two residues per turn and a helical pitch of 5.8 Å. By 2D ROESY experiments, molecular dynamics simulations, and CD spectroscopy we were able to identify the 28 -helix as the predominant conformation in organic solvents. In aqueous solution, the α-aminoxy peptides exist in the 28 -helical conformation at acidic pH, but exhibit remarkable changes in the secondary structure with increasing pH. The most cytotoxic α-aminoxy peptides have an increased propensity to take up a 28 -helical conformation in the presence of a model membrane. This indicates a correlation between the 28 -helical conformation and the membranolytic activity observed in mode of action studies, thereby providing novel insights in the folding properties and the biological activity of α-aminoxy peptides.

Polysaccharide hydrogels with tunable stiffness and provasculogenic properties via α-helix to ß-sheet switch in secondary structure.

Mechanical aspects of the cellular environment can influence cell function, and in this context hydrogels can serve as an instructive matrix. Here we report that physicochemical properties of hydrogels derived from polysaccharides (agarose, κ-carrageenan) having an α-helical backbone can be tailored by inducing a switch in the secondary structure from α-helix to ß-sheet through carboxylation. This enables the gel modulus to be tuned over four orders of magnitude (G' 6 Pa-3.6 × 10(4) Pa) independently of polymer concentration and molecular weight. Using carboxylated agarose gels as a screening platform, we demonstrate that soft-carboxylated agarose provides a unique environment for the polarization of endothelial cells in the presence of soluble and bound signals, which notably does not occur in fibrin and collagen gels. Furthermore, endothelial cells organize into freestanding lumens over 100 µm in length. The finding that a biomaterial can modulate soluble and bound signals provides impetus for exploring mechanobiology paradigms in regenerative therapies.

Solvation-induced helicity inversion of pseudotetrahedral chiral copper(II) complexes.

The helicity of four-coordinated nonplanar complexes is strongly correlated to the chirality of the ligand. However, the stereochemical induction of either the Δ- or the Λ-configuration at the metal ion is also modulated by environmental factors that change the conformational distribution of ligand rotamers. Calculation of the potential energy surface of bis{(R)-N-(1-(4-X-phenyl)ethyl)salicylaldiminato-κ(2)N,O}copper(II) with X = Cl at the density functional theory level showed a clear dependence of the helicity-determining angle θ between the two coordination planes on the relative population of different ligand conformers. The influence of different substituents (X = H, Cl, Br, and OCH3) on complex helicity was studied by determination of the absolute configuration at the metal ion in complexes with either (R)- or (S)-configured ligands. X-ray single-crystal analysis showed that (R)-configured ligands with H, Cl, Br induce Δ, while OCH3-substituted (R)-configured ligands induce Λ in the solid state. According to vibrational circular dichroism and electronic circular dichroism studies in solution, however, all tested complexes with (R)-ligands exhibited a propensity for Δ, with high diastereomeric ratio for X = Cl and X = Br and moderate diastereomeric ratio for X = H and X = OCH3 substituted ligands. Therefore, solvation of copper complexes with X = OCH3 goes along with helicity inversion. This solid-state versus solution study demonstrates that it is not sufficient to determine the chiral-at-metal configuration of a compound by X-ray crystallography alone, because the solution structure can be different. This is particularly important for the use of chiral-at-metal complexes as catalysts in stereoselective synthesis.

Mechanically tailored agarose hydrogels through molecular alloying with ß-sheet polysaccharides.

There is mounting evidence that the mechanical property of tissues provides important cues that control cell fate. However, implementation of hydrogels with tunable physicochemical properties is limited due to the challenges associated with crosslinking chemistries. It has been recently shown that mechanically well-defined injectable polysaccharide hydrogels can be engineered by switching their secondary structure from an α-helix to a ß-sheet. Based on these findings, a new concept is presented to tailor the mechanical properties of agarose hydrogels via the blending with the ß-sheet-rich carboxylated derivative. Using this simple strategy, gels with predictable roughness, fiber organization, and shear modulus ranging from 0.1 to 100 kPa can be formulated. Hydrogels whose mechanical properties can be precisely tailored in vivo without the recourse for chemical reactions are expected to play an important role in implementing mechanobiology paradigms in de novo tissue engineering.

Regio- and stereoselective intermolecular oxidative phenol coupling in Streptomyces.

Intermolecular oxidative phenol coupling is the main process in nature for the formation of atroposelective biaryl compounds. Although well defined in plants and fungi, this type of dimerization reaction in bacteria is poorly understood. Therefore, the biosynthesis of julichromes, spectomycins, and setomimycin was investigated. The monomeric subunits of these biarylic pre-anthraquinones are derived from a common polyketidic precursor, yet the coupling reaction proceeds in a regioselective manner, with the position of attachment of the two subunits depending on the specific streptomycete strain. By using genome analysis and deletion experiments, the biosynthetic gene clusters were identified. Furthermore, it was established that cytochrome P450 enzymes are fundamentally involved during dimerization of the polyketide monomers.

Reaction monitoring using mid-infrared laser-based vibrational circular dichroism.

Changes in vibrational circular dichroism (VCD) were recorded on-line during a chemical reaction. The chiral complex nickel-(-)-sparteine chloride was hydrolyzed to free (-)-sparteine base in a biphasic system of sodium hydroxide solution and chloroform (CHCl(3)). Infrared (IR) and VCD spectra were iteratively recorded after pumping a sample from the CHCl(3) phase through a lab-built VCD spectrometer equipped with a tunable mid-IR quantum cascade laser light source, which allows for VCD measurements even in the presence of strongly absorbing backgrounds. Time-dependent VCD spectra were analyzed by singular value decomposition and global exponential fitting. Spectral features corresponding to the complex and free (-)-sparteine could be clearly identified in the fitted amplitude spectrum, which was associated with an exponential decay with an apparent time constant of 127 min (t(½) = 88 min).

Unprecedented role of hydronaphthoquinone tautomers in biosynthesis.

Quinones and hydroquinones are among the most common cellular cofactors, redox mediators, and natural products. Here, we report on the reduction of 2-hydroxynaphthoquinones to the stable 1,4-diketo tautomeric form of hydronaphthoquinones and their further reduction by fungal tetrahydroxynaphthalene reductase. The very high diastereomeric and enantiomeric excess, together with the high yield of cis-3,4-dihydroxy-1-tetralone, exclude an intermediary hydronaphthoquinone. Labeling experiments with NADPH and NADPD corroborated the formation of an unexpected 1,4-diketo tautomeric form of 2-hydroxyhydronaphthoquinone as a stable intermediate. Similar 1,4-diketo tautomers of hydronaphthoquinones were established as products of the NADPH-dependent enzymatic reduction of other 1,4-naphthoquinones, and as substrates for different members of the superfamily of short-chain dehydrogenases. We propose an essential role of hydroquinone diketo tautomers in biosynthesis and detoxification processes.