Biocatalytic Heteroaromatic Amide Formation in Water Enabled by a Catalytic Tetrad and Two Access Tunnels.

The amide moiety belongs to the most common motives in pharmaceutical chemistry, present in many prescribed small-molecule pharmaceuticals. Methods for its manufacture are still in high demand, especially using water/buffer as a solvent and avoiding stoichiometric amounts of activation reagents. Herein, we identified from a library of lipases/esterases/acyltransferases and variants thereof a lipase originating from Sphingomonas sp. HXN-200 (SpL) able to form amides in aqueous solution starting from a broad scope of sterically demanding heteroaromatic ethyl esters as well as aliphatic amines, reaching isolated yields up to 99% on preparative scale and space time yields of up to 864 g L-1 d-1; thus, in selected cases, the amide was formed within minutes. The enzyme features an aspartate next to the canonical serine of the catalytic triad, which was essential for amide formation. Furthermore, the enzyme structure revealed two tunnels to the active site, presumably one for the ester and one for the amine, which permit the bringing together of the sterically demanding heteroaromatic esters and the amine in the active site. This work shows that biocatalytic amide formation starting from various five- and six-membered heteroaromatic ethyl esters in the buffer can serve as a platform for preparative amide synthesis.

Recovery of Hydroxytyrosol from Olive Mill Wastewater Using the Promiscuous Hydrolase/Acyltransferase PestE.

Olive mill wastewater (OMWW) is produced annually during olive oil extraction and contains most of the health-promoting 3-hydroxytyrosol of the olive fruit. To facilitate its recovery, enzymatic transesterification of hydroxytyrosol (HT) was directly performed in an aqueous system in the presence of ethyl acetate, yielding a 3-hydroxytyrosol acetate rich extract. For this, the promiscuous acyltransferase from Pyrobaculum calidifontis VA1 (PestE) was engineered by rational design. The best mutant for the acetylation of hydroxytyrosol (PestE_I208A_L209F_N288A) was immobilized on EziG2 beads, resulting in hydroxytyrosol conversions between 82 and 89 % in one hour, for at least ten reaction cycles in a buffered hydroxytyrosol solution. Due to inhibition by other phenols in OMWW the conversions of hydroxytyrosol from this source were between 51 and 62 %. In a preparative scale reaction, 13.8 mg (57 %) of 3-hydroxytyrosol acetate was extracted from 60 mL OMWW.

Rational Design for Enhanced Acyltransferase Activity in Water Catalyzed by the Pyrobaculum calidifontis VA1 Esterase.

Biocatalytic transesterification is commonly carried out employing lipases in anhydrous organic solvents since hydrolases usually prefer hydrolysis over acyl transfer in bulk water. However, some promiscuous acyltransferases can catalyze acylation in an aqueous solution. In this study, a rational design was performed to enhance the acyltransferase selectivity and substrate scope of the Pyrobaculum calidifontis VA1 esterase (PestE). PestE wild type and variants were applied for the acylation of monoterpene alcohols. The mutant PestE_I208A is selective for (-)-menthyl acetate (E-Value = 55). Highly active acyltransferases were designed, allowing for complete conversion of (-)-citronellol to citronellyl acetate. Additionally, carvacrol was acetylated but with lower conversions. To the best of our knowledge, this is the first example of the biocatalytic acylation of a phenolic alcohol in bulk water. In addition, a high citronellol conversion of 92% was achieved with the more environmentally friendly and inexpensive acyl donor ethyl acetate using PestE_N288F as a catalyst. PestE_N288F exhibits good acyl transfer activity in an aqueous medium and low hydrolysis activity at the same time. Thus, our study demonstrates an alternative synthetic strategy for acylation of compounds without organic solvents.

Dental tray adhesives and their role as potential transmission medium for microorganisms.

OBJECTIVES: This study aimed to evaluate the possible ability of dental impression tray adhesives to serve as a transmission medium for bacteria and fungi when reusable adhesive applicators are utilized. MATERIALS AND METHODS: Ten flasks with tray adhesive were monitored over a period of 12 weeks during clinical use for contamination with bacteria or fungi. Adhesive fluid samples were cultivated on eight different culture media. All grown colonies were identified by using mass spectrometry (MALDI-TOF). Isolates without reliable identification were either identified by Rapid ID 32 API-STREP V3.0 or by sequencing the 16S rRNA genes. RESULTS: After 4 weeks, bacterial growth was detected on chocolate blood agar plates in five different samples. The bacterial species were identified as Staphylococcus warnerii, Staphylococcus epidermidis, Staphylococcus pasteuri, Ralstonia insidiosa, and Alloiococcus otitidis. After 8 weeks Streptococcus oralis grew on a blood agar plate. In all samples, no fungi were identified. CONCLUSIONS: The disinfectant component of the tested tray adhesive seems to be effective. However, some bacteria survived in the flask for a clinically relevant time, which might result in a potential transmission to a new host.

Discovery and Design of Family†VIII Carboxylesterases as Highly Efficient Acyltransferases.

Promiscuous acyltransferase activity is the ability of certain hydrolases to preferentially catalyze acyl transfer over hydrolysis, even in bulk water. However, poor enantioselectivity, low transfer efficiency, significant product hydrolysis, and limited substrate scope represent considerable drawbacks for their application. By activity-based screening of several hydrolases, we identified the family VIII carboxylesterase, EstCE1, as an unprecedentedly efficient acyltransferase. EstCE1 catalyzes the irreversible amidation and carbamoylation of amines in water, which enabled the synthesis of the drug moclobemide from methyl 4-chlorobenzoate and 4-(2-aminoethyl)morpholine (ca. 20 % conversion). We solved the crystal structure of EstCE1 and detailed structure-function analysis revealed a three-amino acid motif important for promiscuous acyltransferase activity. Introducing this motif into an esterase without acetyltransferase activity transformed a "hydrolase" into an "acyltransferase".

Sequence-Based Prediction of Promiscuous Acyltransferase Activity in Hydrolases.

Certain hydrolases preferentially catalyze acyl transfer over hydrolysis in an aqueous environment. However, the molecular and structural reasons for this phenomenon are still unclear. Herein, we provide evidence that acyltransferase activity in esterases highly correlates with the hydrophobicity of the substrate-binding pocket. A hydrophobicity scoring system developed in this work allows accurate prediction of promiscuous acyltransferase activity solely from the amino acid sequence of the cap domain. This concept was experimentally verified by systematic investigation of several homologous esterases, leading to the discovery of five novel promiscuous acyltransferases. We also developed a simple yet versatile colorimetric assay for rapid characterization of novel acyltransferases. This study demonstrates that promiscuous acyltransferase activity is not as rare as previously thought and provides access to a vast number of novel acyltransferases with diverse substrate specificity and potential applications.

Structure of the plastic-degrading Ideonella sakaiensis MHETase bound to a substrate.

The extreme durability of polyethylene terephthalate (PET) debris has rendered it a long-term environmental burden. At the same time, current recycling efforts still lack sustainability. Two recently discovered bacterial enzymes that specifically degrade PET represent a promising solution. First, Ideonella sakaiensis PETase, a structurally well-characterized consensus α/ß-hydrolase fold enzyme, converts PET to mono-(2-hydroxyethyl) terephthalate (MHET). MHETase, the second key enzyme, hydrolyzes MHET to the PET educts terephthalate and ethylene glycol. Here, we report the crystal structures of active ligand-free MHETase and MHETase bound to a nonhydrolyzable MHET analog. MHETase, which is reminiscent of feruloyl esterases, possesses a classic α/ß-hydrolase domain and a lid domain conferring substrate specificity. In the light of structure-based mapping of the active site, activity assays, mutagenesis studies and a first structure-guided alteration of substrate specificity towards bis-(2-hydroxyethyl) terephthalate (BHET) reported here, we anticipate MHETase to be a valuable resource to further advance enzymatic plastic degradation.

A chemical reaction controlled by light-activated molecular switches based on hetero-cyclopentanediyls.

Molecular switches are molecules that can reversibly be shifted between at least two stable states with different physical and chemical properties, making them interesting for application as chemical sensors or molecular machines. We recently discovered that five-membered, cyclic biradicals based on group 15 elements are efficient and robust photochemical switches that can be activated by red light. The quantum yield of the photo-isomerization is as high as 24.6%, and the thermal equilibration of the photo-activation product proceeds rapidly at ambient temperature. The fully reversible process was studied by experimental and high-level ab initio techniques. We could further demonstrate that the biradical character could be completely turned on and off, so the system could be applied to control chemical equilibria that involve activation products of the cyclic biradicals.

Molecular basis for diversification of yeast prion strain conformation.

Self-propagating ß-sheet-rich fibrillar protein aggregates, amyloid fibers, are often associated with cellular dysfunction and disease. Distinct amyloid conformations dictate different physiological consequences, such as cellular toxicity. However, the origin of the diversity of amyloid conformation remains unknown. Here, we suggest that altered conformational equilibrium in natively disordered monomeric proteins leads to the adaptation of alternate amyloid conformations that have different phenotypic effects. We performed a comprehensive high-resolution structural analysis of Sup35NM, an N-terminal fragment of the Sup35 yeast prion protein, and found that monomeric Sup35NM harbored latent local compact structures despite its overall disordered conformation. When the hidden local microstructures were relaxed by genetic mutations or solvent conditions, Sup35NM adopted a strikingly different amyloid conformation, which redirected chaperone-mediated fiber fragmentation and modulated prion strain phenotypes. Thus, dynamic conformational fluctuations in natively disordered monomeric proteins represent a posttranslational mechanism for diversification of aggregate structures and cellular phenotypes.

Using a map-based assessment tool for the development of cost-effective WFD river basin action programmes in a changing climate.

For the 2nd and 3rd river basin management cycles (2015-2027) of the Water Framework Directive (WFD), EU Member States are required to fully integrate climate change into the process of river basin management planning (RBMP). Complying with the main WFD objective of achieving 'good ecological status' in all water bodies in Denmark requires Programmes of Measures (PoMs) to reduce nitrogen (N) pollution from point and diffuse sources. Denmark is among the world's most intensively farmed countries and in spite of thirty years of significant policy actions to reduce diffuse nutrient emissions, there is still a need for further reductions. In addition, the impacts of climate change are projected to lead to a situation where nutrient loads will have to be reduced still further in comparison to current climate conditions. There is an urgent need to address this challenge in WFD action programmes in order to develop robust and cost-effective adaptation strategies for the next WFD RBMP cycles. The aim of this paper is to demonstrate and discuss how a map-based PoMs assessment tool can support the development of adaptive and cost-effective strategies to reduce N losses in the Isefjord and Roskilde Fjord River Basin in the north east of Denmark. The tool facilitates assessments of the application of agri-environmental measures that are targeted towards low retention agricultural areas, where limited or no surface and subsurface N reduction takes place. Effects of climate change on nitrate leaching were evaluated using the dynamic agro-ecosystem model 'Daisy'. Results show that nitrate leaching rates increase by approx. 25% under current management practices. This impact outweighs the expected total N reduction effect of Baseline 2015 and the first RBMP in the case study river basin. The particular PoMs investigated in our study show that WFD N reduction targets can be achieved by targeted land use changes on approx. 4% of the agricultural area under current climate conditions and approx. 9% of the agricultural area, when projected climate change impacts on nitrate leaching rates are included in the assessment. The study highlights the potential of the PoMs assessment tool to assist in evaluation of alternative WFD RBMP scenarios to achieve spatially targeted and cost-effective reductions of N loads at catchment scale in the context of a changing climate.

Harnessing NMR relaxation interference effects to characterise supramolecular assemblies.

Solution-state NMR spectroscopy remains the primary method for characterising synthetic supramolecular assemblies. Yet, in their NMR spectra, relaxation interference effects can significantly alter peak intensities hindering interpretation. Here, we present a simple experiment for synthetic chemists to analyse this effect, allowing interpretation of these distorted spectra and validation of spectral assignments. We apply this experiment to synthetic porphyrin oligomers with molecular weights approaching those of protein domains (10 kDa). Our experiment provides a simple means to gain additional structural and dynamical information that will become increasingly useful as chemists create larger molecular architectures.

Progress towards structural understanding of infectious sheep PrP-amyloid.

The still elusive structural difference of non-infectious and infectious amyloid of the mammalian prion protein (PrP) is a major pending milestone in understanding protein-mediated infectivity in neurodegenerative diseases. Preparations of PrP-amyloid proven to be infectious have never been investigated with a high-resolution technique. All available models to date have been based on low-resolution data. Here, we establish protocols for the preparation of infectious samples of full-length recombinant (rec) PrP-amyloid in NMR-sufficient amounts by spontaneous fibrillation and seeded fibril growth from brain extract. We link biological and structural data of infectious recPrP-amyloid, derived from bioassays, atomic force microscopy, and solid-state NMR spectroscopy. Our data indicate a semi-mobile N-terminus, some residues with secondary chemical shifts typical of α-helical secondary structure in the middle part between ∼115 to ∼155, and a distinct ß-sheet core C-terminal of residue ∼155. These findings are not in agreement with all current models for PrP-amyloid. We also provide evidence that samples seeded from brain extract may not differ in the overall arrangement of secondary structure elements, but rather in the flexibility of protein segments outside the ß-core region. Taken together, our protocols provide an essential basis for the high-resolution characterization of non-infectious and infectious PrP-amyloid in the near future.

Expression and characterisation of fully posttranslationally modified cellular prion protein in Pichia pastoris.

Prion diseases are fatal neurodegenerative diseases which occur as sporadic, genetic, and transmissible disorders. A molecular hallmark of prion diseases is the conformational conversion of the host-encoded cellular form of the prion protein (PrPC) into its misfolded pathogenic isoform (PrPSc). PrPSc is the main component of the pathological and infectious prion agent. The study of the conversion mechanism from PrPC to PrPSc is a major field in prion research. PrPC is glycosylated and attached to the plasma membrane via its glycosyl phosphatidyl inositol (GPI)-anchor. In this study we established and characterised the expression of fully posttranslationally modified mammalian Syrian golden hamster PrPC in the yeast Pichia pastoris using native PrPC-specific N- and C-terminal signal sequences. In vivo as well as in vitro-studies demonstrated that the signal sequences controlled posttranslational processing and trafficking of native PrPC, resulting in PrPC localised in the plasma membrane of P. pastoris. In addition, the glycosylation pattern of native PrPC could be confirmed.

Solid-state NMR spectroscopy of proteins.

Solid-state NMR spectroscopy proved to be a versatile tool for characterization of structure and dynamics of complex biochemical systems. In particular, magic angle spinning (MAS) solid-state NMR came to maturity for application towards structural elucidation of biological macromolecules. Current challenges in applying solid-state NMR as well as progress achieved recently will be discussed in the following chapter focusing on conceptual aspects important for structural elucidation of proteins.

ß-Sheet core of tau paired helical filaments revealed by solid-state NMR.

One of the hallmarks of Alzheimer's disease is the self-assembly of the microtubule-associated protein tau into fibers termed "paired helical filaments" (PHFs). However, the structural basis of PHF assembly at atomic detail is largely unknown. Here, we applied solid-state nuclear magnetic resonance (ssNMR) spectroscopy to investigate in vitro assembled PHFs from a truncated three-repeat tau isoform (K19) that represents the core of PHFs. We found that the rigid core of the fibrils is formed by amino acids V306 to S324, only 18 out of 99 residues, and comprises three ß-strands connected by two short kinks. The first ß-strand is formed by the well-studied hexapeptide motif VQIVYK that is known to self-aggregate in a steric zipper arrangement. Results on mixed [(15)N:(13)C]-labeled K19 fibrils show that ß-strands are stacked in a parallel, in-register manner. Disulfide bridges formed between C322 residues of different molecules lead to a disturbance of the ß-sheet structure, and polymorphism in ssNMR spectra is observed. In particular, residues K321-S324 exhibit two sets of resonances. Experiments on K19 C322A PHFs further confirm the influence of disulfide bond formation on the core structure. Our structural data are supported by H/D exchange NMR measurements on K19 as well as a truncated four-repeat isoform of tau (K18). Site-directed mutagenesis studies show that single-point mutations within the three different ß-strands result in a significant loss of PHF aggregation efficiency, highlighting the importance of the ß-structure-rich regions for tau aggregation.

MiR-133b targets antiapoptotic genes and enhances death receptor-induced apoptosis.

Despite the importance of microRNAs (miRs) for regulation of the delicate balance between cell proliferation and death, evidence for their specific involvement during death receptor (DR)-mediated apoptosis is scarce. Transfection with miR-133b rendered resistant HeLa cells sensitive to tumor necrosis factor-alpha (TNFα)-induced cell death. Similarly, miR-133b caused exacerbated proapoptotic responses to TNF-related apoptosis-inducing ligand (TRAIL) or an activating antibody to Fas/CD95. Comprehensive analysis, encompassing global RNA or protein expression profiling performed by microarray experiments and pulsed stable isotope labeling with amino acids in cell culture (pSILAC), led to the discovery of the antiapoptotic protein Fas apoptosis inhibitory molecule (FAIM) as immediate miR-133b target. Moreover, miR-133b impaired the expression of the detoxifying protein glutathione-S-transferase pi (GSTP1). Expression of miR-133b in tumor specimens of prostate cancer patients was significantly downregulated in 75% of the cases, when compared with matched healthy tissue. Furthermore, introduction of synthetic miR-133b into an ex-vivo model of prostate cancer resulted in impaired proliferation and cellular metabolic activity. PC3 cells were also sensitized to apoptotic stimuli after transfection with miR-133b similar to HeLa cells. These data reveal the ability of a single miR to influence major apoptosis pathways, suggesting an essential role for this molecule during cellular transformation, tumorigenesis and tissue homeostasis.

Structure-activity relationships of flavonoids as inhibitors of breast cancer resistance protein (BCRP).

Flavonoids are an interesting group of natural products ubiquitously present in human diet. Their consumption has been associated with various and differing beneficial health effects. However, several flavonoids have been reported to inhibit the breast cancer resistance protein (BCRP) encoded by the ABCG2 gene. Thus, the consumption of flavonoids with high inhibitory activity could change pharmacokinetics and drug levels of drugs that are BCRP substrates. In cancer patients receiving chemotherapy an increased intake of such flavonoids could lead to adverse effects. We investigated a structurally diverse set of flavonoids, including derivatives with a rare C-methylated structure that were isolated from plants used in traditional medicine. The flavones retusin and ayanin were found to be highly potent inhibitors of BCRP, showing only slightly less potency than Ko143, the most potent ABCG2 inhibitor known so far. The activity data were analyzed by 2D and 3D QSAR analyses and the results revealed the impact of the different substituents at the various positions of the flavonoid core on activity. Additionally, a lateral 2D QSAR analysis of data collected from the literature was performed aiming to derive more general information about the influence of distinct structural features on the inhibitory potency of flavonoids. The comparative QSAR analyses led to a consistent picture of the effects of the different substituents at various positions of the flavone backbone. The following structural features were found to contribute positively to BCRP inhibition: a hydroxyl group in position 5, double bond between position 2 and 3, and a methoxy group in position 3. The exchange of a 3-methoxy group by an OH-group acting also as a hydrogen bond donor, resulted in decrease in activity underlining the potential role of the hydrogen bond acceptor 3-OCH(3) for the interaction with BCRP.

Total synthesis of natural and non-natural Δ(5,6)Δ(12,13)-jatrophane diterpenes and their evaluation as MDR modulators.

We report the details of the total synthesis of natural and non-natural jatropha-5,12-dienes. The successful tactic for the assembly of the strained trans-bicyclo[10.3.0]pentadecane scaffold employed a B-alkyl Suzuki-Miyaura cross-coupling for the formation of the C5/C6 double bond and a ring-closing metathesis for the construction of the C12/C13 double bond. The key step of the synthesis of the cyclopentane fragment, an uncatalyzed intramolecular carbonyl-ene reaction, was studied computationally by DFT calculations. The members of the ensemble of synthetic natural and non-natural jatrophanes were subsequently examined as modulators for the ABCB1, ABCG2, and ABCC1 efflux proteins, which are associated with multidrug resistance in cancer chemotherapy.

Novel lead for potent inhibitors of breast cancer resistance protein (BCRP).

From our modulator library an interesting inhibitor of breast cancer resistance protein (BCRP) was identified. Due to its high inhibitory potency, this compound may serve as a promising novel lead for the design of new and potent modulators. This adds a new structural class to the few known highly active BCRP inhibitors.

Synthesis and biological evaluation of a small molecule library of 3rd generation multidrug resistance modulators.

The development of new modulators possessing high efficacy, low toxicity and high selectivity is a pivotal approach to overcoming P-glycoprotein (P-gp) mediated multidrug resistance (MDR) in tumour cells. In this study 39 compounds are presented which have been synthesized and pharmacologically investigated in our laboratory. Similarly to the potent 3rd generation MDR modulator tariquidar (XR9576) the compounds contain a tetrahydroisoquinoline-ethyl-phenylamine substructure that, in contrast to XR9576, is connected to a smaller hydrophobic part, thus leading to molecules of lower molecular weight. The connection between the tetrahydroisoquinoline-ethyl-phenylamine substructure and the hydrophobic part was achieved through four different types of linkers: amide, urea, amide-ether and amide-styryl. A number of structural modifications in the hydrophobic part were created. The calcein AM assay served as test system to determine the P-gp transport inhibitory potencies of the compounds. For the amide linker derivatives a structure-activity relationship analysis was performed outlining which structural modifications contributed to the inhibitory potency. The compounds containing a bicyclic hydrophobic part with a particular substituent in a specific orientation were identified as the most potent amide derivatives. Among the urea derivatives the compounds with highest inhibitory potency possessed an ortho-nitro substituent. The conformational analysis revealed that this position enables the formation of a hydrogen bond to the urea linker thus stabilizing the conformation. Regarding the amide-styryl derivatives the elongation of the amide linker seemed to be most decisive for the observed increase in activity. The most promising candidate in the whole library possess an amide-ether linker and an ortho-nitro substituent in the hydrophobic part. This compound inhibites P-gp slightly less than tariquidar and can serve as a lead structure for new potent P-gp modulators.