Dynamic Heterogeneity of Filler-Associated Interphases in Polymer Nanocomposites.

Dynamically inhomogeneous polymer systems exhibit interphases with mobility gradients. These are believed to play key roles in the material's performance. A prominent example is particle-filled rubber, a special case of a crosslinked polymer nanocomposite, where favorable rubber-filler interactions may give rise to a nanoscale immobilized layer around the filler, including regions of intermediate mobility. Such intermediate domains may either form a separate shell-like layer or be a manifestation of dynamic heterogeneities, in which case the intermediately mobile material would be dispersed in the form of nanometer-sized subdomains. In this contribution, bidirectional proton NMR spin diffusion (SD) experiments applied to silica-filled acrylate rubber are combined with numerical simulations to provide microscopic insights into this question. While model calculations for different scenarios fit the given data similarly well for longer SD mixing time, the short-time data do support the presence of dynamic heterogeneities.

Expanding the Toolbox of R-Selective Amine Transaminases by Identification and Characterization of New Members.

Amine transaminases (ATAs) are used to synthesize enantiomerically pure amines, which are building blocks for pharmaceuticals and agrochemicals. R-selective ATAs belong to the fold type IV PLP-dependent enzymes, and different sequence-, structure- and substrate scope-based features have been identified in the past decade. However, our knowledge is still restricted due to the limited number of characterized (R)-ATAs, with additional bias towards fungal origin. We aimed to expand the toolbox of (R)-ATAs and contribute to the understanding of this enzyme subfamily. We identified and characterized four new (R)-ATAs. The ATA from Exophiala sideris contains a motif characteristic for d-ATAs, which was previously believed to be a disqualifying factor for (R)-ATA activity. The crystal structure of the ATA from Shinella is the first from a Gram-negative bacterium. The ATAs from Pseudonocardia acaciae and Tetrasphaera japonica are the first characterized (R)-ATAs with a shortened/missing N-terminal helix. The active-site charges vary significantly between the new and known ATAs, correlating with their diverging substrate scope.

Functional Expression and Characterization of a Panel of Cobalt and Iron-Dependent Nitrile Hydratases.

Nitrile hydratases (NHase) catalyze the hydration of nitriles to the corresponding amides. We report on the heterologous expression of various nitrile hydratases. Some of these enzymes have been investigated by others and us before, but sixteen target proteins represent novel sequences. Of 21 target sequences, 4 iron and 16 cobalt containing proteins were functionally expressed from Escherichia coli BL21 (DE3) Gold. Cell free extracts were used for activity profiling and basic characterization of the NHases using the typical NHase substrate methacrylonitrile. Co-type NHases are more tolerant to high pH than Fe-type NHases. A screening for activity on three structurally diverse nitriles was carried out. Two novel Co-dependent NHases from Afipia broomeae and Roseobacter sp. and a new Fe-type NHase from Gordonia hydrophobica were very well expressed and hydrated methacrylonitrile, pyrazine-carbonitrile, and 3-amino-3-(p-toluoyl)propanenitrile. The Co-dependent NHases from Caballeronia jiangsuensis and Microvirga lotononidis, as well as two Fe-dependent NHases from Pseudomonades, were-in addition-able to produce the amide from cinnamonitrile. Summarizing, seven so far uncharacterized NHases are described to be promising biocatalysts.

Enzymes revolutionize the bioproduction of value-added compounds: From enzyme discovery to special applications.

Competitive sustainable production in industry demands new and better biocatalysts, optimized bioprocesses and cost-effective product recovery. Our review sheds light on the progress made for the individual steps towards these goals, starting with the discovery of new enzymes and their corresponding genes. The enzymes are subsequently engineered to improve their performance, combined in reaction cascades to expand the reaction scope and integrated in whole cells to provide an optimal environment for the bioconversion. Strain engineering using synthetic biology methods tunes the host for production, reaction design optimizes the reaction conditions and downstream processing ensures the efficient recovery of commercially viable products. Selected examples illustrate how modified enzymes can revolutionize future-oriented applications ranging from the bioproduction of bulk-, specialty- and fine chemicals, active pharmaceutical ingredients and carbohydrates, over the conversion of the greenhouse-gas CO2 into valuable products and biocontrol in agriculture, to recycling of synthetic polymers and recovery of precious metals.

Production of Hydroxynitrile Lyase from Davallia tyermannii (DtHNL) in Komagataella phaffii and Its Immobilization as a CLEA to Generate a Robust Biocatalyst.

Hydroxynitrile lyase from the white rabbit's foot fern Davallia tyermannii (DtHNL) catalyzes the enantioselective synthesis of α-cyanohydrins, which are key building blocks for pharmaceutical and agrochemical industries. An efficient and competitive process necessitates the availability and robustness of the biocatalyst. Herein, the recombinant production of DtHNL1 in Komagataella phaffii, yielding approximately 900 000 U L-1 , is described. DtHNL1 constitutes approximately 80 % of the total protein content. The crude enzyme was immobilized. Crosslinked enzyme aggregates (CLEAs) resulted in significant enhancement of the biocatalyst stability under acidic conditions (activity retained after 168 h at pH 2.4). The DtHNL1-CLEA was employed for (R)-mandelonitrile synthesis (99 % conversion, 98 % enantiomeric excess) in a biphasic system, and evaluated for the synthesis of (R)-hydroxypivaldehyde cyanohydrin under reaction conditions that immediately inactivated non-immobilized DtHNL1. The results show the DtHNL1-CLEA to be a stable biocatalyst for the synthesis of enantiomerically pure cyanohydrins under acidic conditions.

Enzyme discovery beyond homology: a unique hydroxynitrile lyase in the Bet v1 superfamily.

Homology and similarity based approaches are most widely used for the identification of new enzymes for biocatalysis. However, they are not suitable to find truly novel scaffolds with a desired function and this averts options and diversity. Hydroxynitrile lyases (HNLs) are an example of non-homologous isofunctional enzymes for the synthesis of chiral cyanohydrins. Due to their convergent evolution, finding new representatives is challenging. Here we show the discovery of unique HNL enzymes from the fern Davallia tyermannii by coalescence of transcriptomics, proteomics and enzymatic screening. It is the first protein with a Bet v1-like protein fold exhibiting HNL activity, and has a new catalytic center, as shown by protein crystallography. Biochemical properties of D. tyermannii HNLs open perspectives for the development of a complementary class of biocatalysts for the stereoselective synthesis of cyanohydrins. This work shows that systematic integration of -omics data facilitates discovery of enzymes with unpredictable sequences and helps to extend our knowledge about enzyme diversity.

Erratum: Enzyme discovery beyond homology: a unique hydroxynitrile lyase in the Bet v1 superfamily.

This corrects the article DOI: 10.1038/srep46738.

Crystal Structure and Catalytic Mechanism of CouO, a Versatile C-Methyltransferase from Streptomyces rishiriensis.

Friedel-Crafts alkylation of aromatic systems is a classic reaction in organic chemistry, for which regiospecific mono-alkylation, however, is generally difficult to achieve. In nature, methyltransferases catalyze the addition of methyl groups to a wide range of biomolecules thereby modulating the physico-chemical properties of these compounds. Specifically, S-adenosyl-L-methionine dependent C-methyltransferases possess a high potential to serve as biocatalysts in environmentally benign organic syntheses. Here, we report on the high resolution crystal structure of CouO, a C-methyltransferase from Streptomyces rishiriensis involved in the biosynthesis of the antibiotic coumermycin A1. Through molecular docking calculations, site-directed mutagenesis and the comparison with homologous enzymes we identified His120 and Arg121 as key functional residues for the enzymatic activity of this group of C-methyltransferases. The elucidation of the atomic structure and the insight into the catalytic mechanism provide the basis for the (semi)-rational engineering of the enzyme in order to increase the substrate scope as well as to facilitate the acceptance of SAM-analogues as alternative cofactors.

Discovery and structural characterisation of new fold type IV-transaminases exemplify the diversity of this enzyme fold.

Transaminases are useful biocatalysts for the production of amino acids and chiral amines as intermediates for a broad range of drugs and fine chemicals. Here, we describe the discovery and characterisation of new transaminases from microorganisms which were enriched in selective media containing (R)-amines as sole nitrogen source. While most of the candidate proteins were clearly assigned to known subgroups of the fold IV family of PLP-dependent enzymes by sequence analysis and characterisation of their substrate specificity, some of them did not fit to any of these groups. The structure of one of these enzymes from Curtobacterium pusillum, which can convert d-amino acids and various (R)-amines with high enantioselectivity, was solved at a resolution of 2.4 Å. It shows significant differences especially in the active site compared to other transaminases of the fold IV family and thus indicates the existence of a new subgroup within this family. Although the discovered transaminases were not able to convert ketones in a reasonable time frame, overall, the enrichment-based approach was successful, as we identified two amine transaminases, which convert (R)-amines with high enantioselectivity, and can be used for a kinetic resolution of 1-phenylethylamine and analogues to obtain the (S)-amines with e.e.s >99%.

Engineering of TM1459 from Thermotoga maritima for Increased Oxidative Alkene Cleavage Activity.

Oxidative cleavage of alkenes is a widely employed process allowing oxyfunctionalization to corresponding carbonyl compounds. Recently, a novel biocatalytic oxidative alkene cleavage activity on styrene derivatives was identified in TM1459 from Thermotoga maritima. In this work we engineered the enzyme by site-saturation mutagenesis of active site amino acids to increase its activity and to broaden its substrate scope. A high-throughput assay for the detection of the ketone products was successfully developed. Several variants with up to twofold improved conversion level of styrene derivatives were successfully identified. Especially, changes in or removal of the C-terminus of TM1459 increased the activity most significantly. These best variants also displayed a slightly enlarged substrate scope.

Amides in Nature and Biocatalysis.

Amides are widespread in biologically active compounds with a broad range of applications in biotechnology, agriculture and medicine. Therefore, as alternative to chemical synthesis the biocatalytic amide synthesis is a very interesting field of research. As usual, Nature can serve as guide in the quest for novel biocatalysts. Several mechanisms for carboxylate activation involving mainly acyl-adenylate, acyl-phosphate or acyl-enzyme intermediates have been discovered, but also completely different pathways to amides are found. In addition to ribosomes, selected enzymes of almost all main enzyme classes are able to synthesize amides. In this review we give an overview about amide synthesis in Nature, as well as biotechnological applications of these enzymes. Moreover, several examples of biocatalytic amide synthesis are given.

Discovery of a novel (R)-selective bacterial hydroxynitrile lyase from Acidobacterium capsulatum.

Hydroxynitrile lyases (HNLs) are powerful carbon-carbon bond forming enzymes. The reverse of their natural reaction - the stereoselective addition of hydrogen cyanide (HCN) to carbonyls - yields chiral cyanohydrins, versatile building blocks for the pharmaceutical and chemical industry. Recently, bacterial HNLs have been discovered, which represent a completely new type: HNLs with a cupin fold. Due to various benefits of cupins (e.g. high yield recombinant expression in Escherichia coli), the class of cupin HNLs provides a new source for interesting, powerful hydroxynitrile lyases in the ongoing search for HNLs with improved activity, enantioselectivity, stability and substrate scope. In this study, database mining revealed a novel cupin HNL from Acidobacterium capsulatum ATCC 51196 (AcHNL), which was able to catalyse the (R)-selective synthesis of mandelonitrile with significantly better conversion (97%) and enantioselectivity (96.7%) than other cupin HNLs.

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.

Crystal structure of an (R)-selective ω-transaminase from Aspergillus terreus.

Chiral amines are important building blocks for the synthesis of pharmaceutical products, fine chemicals, and agrochemicals. ω-Transaminases are able to directly synthesize enantiopure chiral amines by catalysing the transfer of an amino group from a primary amino donor to a carbonyl acceptor with pyridoxal 5'-phosphate (PLP) as cofactor. In nature, (S)-selective amine transaminases are more abundant than the (R)-selective enzymes, and therefore more information concerning their structures is available. Here, we present the crystal structure of an (R)-ω-transaminase from Aspergillus terreus determined by X-ray crystallography at a resolution of 1.6 Å. The structure of the protein is a homodimer that displays the typical class IV fold of PLP-dependent aminotransferases. The PLP-cofactor observed in the structure is present in two states (i) covalently bound to the active site lysine (the internal aldimine form) and (ii) as substrate/product adduct (the external aldimine form) and free lysine. Docking studies revealed that (R)-transaminases follow a dual binding mode, in which the large binding pocket can harbour the bulky substituent of the amine or ketone substrate and the α-carboxylate of pyruvate or amino acids, and the small binding pocket accommodates the smaller substituent.

Mini-review: recent developments in hydroxynitrile lyases for industrial biotechnology.

Hydroxynitrile lyases (HNLs) catalyze the cleavage as well as the formation of cyanohydrins. The latter reaction is valuable for the stereoselective C-C bond formation by condensation of HCN with carbonyl compounds. The resulting cyanohydrins serve as versatile building blocks for a broad range of chemical and enzymatic follow-up reactions. A significant number of (R)- and (S)-selective HNLs are known today and the number is still increasing. HNLs not only exhibit varying substrate scope but also differ in sequence and structure. Tailor-made enzymes for large-scale manufacturing of cyanohydrins with improved yield and enantiomeric excess are very interesting targets, which is reflected in a solid number of patents. This review will complement and extend our recent review with a strong focus on applications of HNLs for the synthesis of highly functionalized, chiral compounds with newest literature, recent and current patent literature.

Biochemical and structural characterization of a novel bacterial manganese-dependent hydroxynitrile lyase.

Hydroxynitrile lyases (HNLs), which catalyse the decomposition of cyanohydrins, are found mainly in plants. In vitro, they are able to catalyse the synthesis of enantiopure cyanohydrins, which are versatile building blocks in the chemical industry. Recently, HNLs have also been discovered in bacteria. Here, we report on the detailed biochemical and structural characterization of a hydroxynitrile lyase from Granulicella tundricola (GtHNL), which was successfully heterologously expressed in Escherichia coli. The crystal structure was solved at a crystallographic resolution of 2.5 Å and exhibits a cupin fold. As GtHNL does not show any sequence or structural similarity to any other HNL and does not contain conserved motifs typical of HNLs, cupins represent a new class of HNLs. GtHNL is metal-dependent, as confirmed by inductively coupled plasma/optical emission spectroscopy, and in the crystal structure, manganese is bound to three histidine and one glutamine residue. GtHNL displayed a specific activity of 1.74 U·mg(-1) at pH 6 with (R)-mandelonitrile, and synthesized (R)-mandelonitrile with 90% enantiomeric excess at 80% conversion using 0.5 m benzaldehyde in a biphasic reaction system with methyl tertiary butyl ether.

Crystallization of a novel metal-containing cupin from Acidobacterium sp. and preliminary diffraction data analysis.

Recombinant AciX9_0562 from Acidobacterium sp. MP5ACTX9 (UniProt ID E8WYN5) containing sequence motifs characteristic of the RmlC-type cupins superfamily and containing Pfam motif PF07883 has been successfully cloned, expressed and purified. AciX9_0562 crystallized in a number of conditions from the Morpheus protein crystallization screen. The best crystal diffracted to 2.7 Å resolution (space group C222(1); unit-cell parameters a = 125.29, b = 254.63, c = 82.99 Å). Structure solution was facilitated by the automated molecular-replacement pipeline BALBES. The initial solution was automatically rebuilt using the PHENIX AutoBuild wizard, with final R and R(free) values of 0.23 and 0.26, respectively. The structure is currently undergoing manual refinement.

Characterization of two bacterial hydroxynitrile lyases with high similarity to cupin superfamily proteins.

Hydroxynitrile lyases (HNLs) catalyze the cleavage of cyanohydrins. In the reverse reaction, they catalyze the formation of carbon-carbon bonds by enantioselective condensation of hydrocyanic acid with carbonyls. In this study, we describe two proteins from endophytic bacteria that display activity in the cleavage and the synthesis reaction of (R)-mandelonitrile with up to 74% conversion of benzaldehyde (enantiopreference ee 89%). Both showed high similarity to proteins of the cupin superfamily which so far were not known to exhibit HNL activity.

Recent advances in rational approaches for enzyme engineering.

Enzymes are an attractive alternative in the asymmetric syntheses of chiral building blocks. To meet the requirements of industrial biotechnology and to introduce new functionalities, the enzymes need to be optimized by protein engineering. This article specifically reviews rational approaches for enzyme engineering and de novo enzyme design involving structure-based approaches developed in recent years for improvement of the enzymes' performance, broadened substrate range, and creation of novel functionalities to obtain products with high added value for industrial applications.

The s-layer glycome-adding to the sugar coat of bacteria.

The amazing repertoire of glycoconjugates present on bacterial cell surfaces includes lipopolysaccharides, capsular polysaccharides, lipooligosaccharides, exopolysaccharides, and glycoproteins. While the former are constituents of Gram-negative cells, we review here the cell surface S-layer glycoproteins of Gram-positive bacteria. S-layer glycoproteins have the unique feature of self-assembling into 2D lattices providing a display matrix for glycans with periodicity at the nanometer scale. Typically, bacterial S-layer glycans are O-glycosidically linked to serine, threonine, or tyrosine residues, and they rely on a much wider variety of constituents, glycosidic linkage types, and structures than their eukaryotic counterparts. As the S-layer glycome of several bacteria is unravelling, a picture of how S-layer glycoproteins are biosynthesized is evolving. X-ray crystallography experiments allowed first insights into the catalysis mechanism of selected enzymes. In the future, it will be exciting to fully exploit the S-layer glycome for glycoengineering purposes and to link it to the bacterial interactome.