Dissolution state of cellulose in aqueous systems. 1. Alkaline solvents

The understanding of the state of dissolution of cellulose in a certain solvent is a critical step forward in the development of new efficient solvent systems for cellulose. Nevertheless, obtaining such information is not trivial. Recently, polarization transfer solid-state NMR (PTssNMR) was shown to be a very promising technique regarding an efficient and robust characterization of the solution state of cellulose. In the present study, combining PTssNMR, microscopic techniques and X-ray diffraction, a set of alkaline aqueous systems are investigated. The addition of specific additives, such as urea or thiourea, to aqueous NaOH based systems as well as the use of an amphiphilic organic cation, is found to have pronounced effects on the dissolution efficiency of cellulose. Additionally, the characteristics of the regenerated material are strongly dependent on the dissolution system; typically less crystalline materials, presenting smoother morphologies, are obtained when amphiphilic solvents or additives are used.     

[FeFe]‐Hydrogenase with Chalcogenide Substitutions at the H‐Cluster Maintains Full H2 Evolution Activity

The [FeFe]‐hydrogenase HYDA1 from Chlamydomonas reinhardtii is particularly amenable to biochemical and biophysical characterization because the H‐cluster in the active site is the only inorganic cofactor present. Herein, we present the complete chemical incorporation of the H‐cluster into the HYDA1‐apoprotein scaffold and, furthermore, the successful replacement of sulfur in the native [4Fe_H] cluster with selenium. The crystal structure of the reconstituted pre‐mature HYDA1[4Fe4Se]_H protein was determined, and a catalytically intact artificial H‐cluster variant was generated upon in vitro maturation. Full hydrogen evolution activity as well as native‐like composition and behavior of the redesigned enzyme were verified through kinetic assays, FTIR spectroscopy, and X‐ray structure analysis. These findings reveal that even a bioinorganic active site with exceptional complexity can exhibit a surprising level of compositional plasticity.     

Characterization of a cold-active and salt tolerant esterase identified by functional screening of Arctic metagenomic libraries

Background

The use of metagenomics in enzyme discovery constitutes a powerful approach to access to genomes of unculturable community of microorganisms and isolate novel valuable biocatalysts for use in a wide range of biotechnological and pharmaceutical fields.

Results

Here we present a novel esterase gene (lip3) identified by functional screening of three fosmid metagenomic libraries, constructed from three marine sediment samples. The sequenced positive fosmid revealed an enzyme of 281 amino acids with similarity to class 3 lipases. The 3D modeling of Lip3 was generated by homology modeling on the basis of four lipases templates [PDB ID: 3O0D, 3NGM, 3G7N, 2QUB] to unravel structural features of this novel enzyme. The catalytic triad of Lip3 was predicted to be Asp207, His267 and the catalytic nucleophile Ser150 in a conserved pentapeptide (GXSXG). The 3D model highlighted the presence of a one-helix lid able to regulate the access of the substrate to the active site when the enzyme binds a hydrophobic interface. Moreover an analysis of the external surface of Lip3 model showed that the majority of the surface regions were hydrophobic (59.6 %) compared with homologous lipases (around 35 %) used as templates. The recombinant Lip3 esterase, expressed and purified from Escherichia coli, preferentially hydrolyzed short and medium length p-nitrophenyl esters with the best substrate being p-nitrophenyl acetate. Further characterization revealed a temperature optimum of 35 °C and a pH optimum of 8.0. Lip3 exhibits a broad temperature stability range and tolerates the presence of DTT, EDTA, PMSF, β-mercaptoethanol and high concentrations of salt. The enzyme was also highly activated by NaCl.

Conclusions

The biochemical characterization and homology model reveals a novel esterase originating from the marine Arctic metagenomics libraries with features of a cold-active, relatively thermostable and highly halotolerant enzyme. Taken together, these results suggest that this esterase could be a highly valuable candidate for biotechnological applications such as organic synthesis reactions and cheese ripening processes.

     

Expanding the Landscape of Diterpene Structural Diversity through Stereochemically Controlled Combinatorial Biosynthesis

Plant‐derived diterpenoids serve as important pharmaceuticals, food additives, and fragrances, yet their low natural abundance and high structural complexity limits their broader industrial utilization. By mimicking the modularity of diterpene biosynthesis in plants, we constructed 51 functional combinations of class I and II diterpene synthases, 41 of which are “new‐to‐nature”. Stereoselective biosynthesis of over 50 diterpene skeletons was demonstrated, including natural variants and novel enantiomeric or diastereomeric counterparts. Scalable biotechnological production for four industrially relevant targets was accomplished in engineered strains of Saccharomyces cerevisiae.     

Screening the molecular surface of human anticoagulant protein C: A search for interaction sites

Protein C (PC), a 62 kDa multi-modular zymogen, is activated to an anticoagulant serine protease (activated PC or APC) by thrombin bound to thrombomodulin on the surface of endothelial cells. PC/APC interacts with many proteins and the characterisation of these interactions is not trivial. However, molecular modelling methods help to study these complex biological processes and provide basis for rational experimental design and interpretation of the results. PC/APC consists of a Gla domain followed by two EGF modules and a serine protease domain. In this report, we present two structural models for full-length APC and two equivalent models for full-length PC, based on the X-ray structures of Gla-domainless APC and of known serine protease zymogens. The overall elongated shape of the models is further cross-validated using size exclusion chromatography which allows evaluation of the Stokes radius (r_s for PC = 33.15 Å r_s for APC = 34.19 Å), frictional ratio and axial ratio. We then propose potential binding sites at the surface of PC/APC using surface hydrophobicity as a determinant of the preferred sites of intermolecular recognition. Most of the predicted binding sites are consistent with previously reported experimental data, while some clusters highlight new regions that should be involved in protein-protein interactions.     

Overall approaches and experiences of first-time participants in the Nuclear Forensics International Technical Working Group’s Fourth Collaborative Material Exercise (CMX-4)

Journal of Radioanalytical and Nuclear Chemistry - The Fourth Collaborative Material Exercise (CMX-4) of the Nuclear Forensics International Technical Working Group (ITWG) registered the largest...     

Density matrix averaged atomic natural orbital (ANO) basis sets for correlated molecular wave functions

Summary Generally contracted basis sets for first row atoms have been constructed using the Atomic Natural Orbital (ANO) approach, with modifications for allowing symmetry breaking and state averaging. The ANOs are constructed by averaging over several atomic states, positive and negative ions, and atoms in an external electric field. The contracted basis sets give virtually identical results as the corresponding uncontracted sets for the atomic properties, which they have been designed to reproduce. The design objective has been to describe the ionization potential, the electron affinity, and the polarizability as accurately as possible. The result is a set of well-balanced basis sets for molecular calculations. The starting primitive sets are 8s4p3d for hydrogen, 9s4p3d for helium, and 14s9p4d3f for the heavier first row atoms.     

Identification of transformation products from β‐blocking agents formed in wetland microcosms using LC‐Q‐ToF

Identification of degradation products from trace organic compounds, which may retain the biological activity of the parent compound, is an important step in understanding the long‐term effects of these compounds on the environment. Constructed wetlands have been successfully utilized to remove contaminants from wastewater effluent, including pharmacologically active compounds. However, relatively little is known about the transformation products formed during wetland treatment. In this study, three different wetland microcosm treatments were used to determine the biotransformation products of the β‐adrenoreceptor antagonists atenolol, metoprolol and propranolol. LC/ESI‐Q‐ToF run in the MS^E and MS/MS modes was used to identify and characterize the degradation products through the accurate masses of precursor and product ions. The results were compared with those of a reference standard when available. Several compounds not previously described as biotransformation products produced in wetlands were identified, including propranolol‐O‐sulfate, 1‐naphthol and the human metabolite N‐deaminated metoprolol. Transformation pathways were significantly affected by microcosm conditions and differed between compounds, despite the compounds' structural similarities. Altogether, a diverse range of transformation products in wetland microcosms were identified and elucidated using high resolving MS. This work shows that transformation products are not always easily predicted, nor formed via the same pathways even for structurally similar compounds. Copyright © 2016 John Wiley & Sons, Ltd.     

Proton Affinities of Cationic Carbone Adducts [AC(PPh3)2]+ (A=Halogen,Hydrogen, Methyl) and Unusual Electronic Structures of the Cations and Dications [AC(H)(PPh3)2]2+

This work reports the syntheses and the first crystal structures of the cationic carbone adducts [FC(PPh₃)₂]⁺ and [BrC(PPh₃)₂]⁺ and the protonated dication [FC(H)(PPh₃)₂]²⁺, which are derived from the carbone C(PPh₃)₂. Quantum chemical calculations and bonding analyses were carried out for the series of cations [AC(PPh₃)₂]⁺ and dications [AC(H)(PPh₃)₂]²⁺, where A=H, Me, F, Cl, Br, I. The bonding analysis suggests that the cations are best described as phosphane complexes L→(CA)⁺←L (L=PPh₃), which are related to the neutral borylene adducts L→(BA)←L (L=cyclic carbene; A=H, aryl) that were recently isolated. The carbone adducts [AC(PPh₃)₂]⁺ possess a π electron lone pair at carbon and they can easily be protonated to the dications [AC(H)(PPh₃)₂]²⁺. The calculations of the dications indicate that the molecules are best represented as complexes L→(CHA)²⁺←L (L=PPh₃) where a carbene dication is stabilized by the ligands. The central carbon atom in the cations and even in the dications carries a negative partial charge, which is larger than the negative charge at fluorine. There is also the peculiar situation in which the carbon–fluorine bonds in [FC(PPh₃)₂]⁺ and [FC(H)(PPh₃)₂]²⁺ exhibit the expected polarity with the negative end at fluorine, but the carbon atom has a larger negative charge than fluorine. Given the similarity of carbodiphosphorane C(PPh₃)₂ and carbodicarbene C(NHC)₂, we expect that analogous compounds [AC(NHC)₂]⁺ and [AC(H)(NHC)₂]²⁺ with similar features as [AC(PPh₃)₂]⁺ and [AC(H)(PPh₃)₂]²⁺ can be isolated.     

Mass‐Selected Circular Dichroism of Supersonic‐Beam‐Cooled [D4]‐(R)‐(+)‐3‐Methylcyclopentanone

UV spectroscopy and electronic circular dichroism (ECD) experiments on supersonic‐beam‐cooled deuterated (R)‐(+)‐3‐methylcyclopentanone ([D₄]‐(R)‐(+)‐3‐MCP) have been performed by using a laser mass spectrometer. The spectral resolution not only allowed excitation and CD measurements for single vibronic transitions but also for the rotational P, Q, and R branches of these transitions. The investigated transition showed the largest anisotropy factor ever observed for chiral molecules in the gas phase, which, due to residual saturation of the excited transition, represents only a lower limit for the real anisotropy factor. Furthermore, one‐color (1+1+1) and two‐color (1+1′) resonance‐enhanced multiphoton ionization (REMPI) measurements were performed and the effusive‐beam (room temperature) and supersonic‐beam results for [D₄]‐(R)‐(+)‐3‐MCP were compared. These results allowed a differentiation between single‐step ECD (comparable to conventional ECD) and cumulative ECD (only possible in multiphoton excitation) under supersonic‐beam conditions.