A spectrophotometric assay for the detection of fungal peroxygenases

Rapid and simple spectrophotometric methods are required for the unambiguous detection of recently discovered fungal peroxygenases in vivo and in vitro. This paper describes a peroxygenase-specific assay using 5-nitro-1,3-benzodioxole as substrate. The product, 4-nitrocatechol, produces a yellow color at pH 7, which can be followed over time at 425 nm (ε(425)=9,700 M(-1) cm(-1)), and a red color when adjusted to pH >12, which can be measured in form of an end-point determination at 514 nm (ε(514)=11,400 M(-1) cm(-1)). The assay is suitable for detecting peroxygenase activities in complex growth media and environmental samples as well as for high-throughput screenings.     

Conversion of polycyclic aromatic hydrocarbons,methyl naphthalenes and dibenzofuran by two fungal peroxygenases

The aim of this work has been to study the substrate specificity of two aromatic peroxygenases concerning polyaromatic compounds of different size and structure as well as to identify the key metabolites of their oxidation. Thus, we report here on new pathways and reactions for 2-methylnaphthalene, 1-methylnaphthalene, dibenzofuran, fluorene, phenanthrene, anthracene and pyrene catalyzed by peroxygenases from Agrocybe aegerita and Coprinellus radians (abbreviated as AaP and CrP). AaP hydroxylated the aromatic rings of all substrates tested at different positions, whereas CrP showed a limited capacity for aromatic ring-hydroxylation and did not hydroxylate phenanthrene but preferably oxygenated fluorene at the non-aromatic C₉-carbon and methylnaphthalenes at the side chain. The results demonstrate for the first time the broad substrate specificity of fungal peroxygenases for polyaromatic compounds, and they are discussed in terms of their biocatalytic and environmental implications.     

Surfing the actomyosin wave: polarization of the C. elegans zygote

Many cell types rely on asymmetrically localized PAR proteins to become polarized. New evidence reveals that cortical flows powered by actomyosin contractions can mobilize PAR complexes to create distinct cortical domains.     

Surfing the third wave of whiteness studies: reflections on Twine and Gallagher

The influential special issue and overview essay co-edited and co-authored respectively, by Winddance Twine and Charles Gallagher, set out their interpretation of whiteness studies’ genealogy, development and future. In this essay I identify their arguments and critique them in the light of a further eight years’ work on the racialization of white identities produced by the global academy. Particular attention is paid to the proliferation of micro studies about an ever-increasing array of sites, both in thematic and international terms, and to the corpus’ addressing of power relations. Moreover, I underscore Twine and Gallagher’s prescience on the strand of the work they review that bears on the racial project of recuperating white supremacy in a variety of ways, a project that is enjoying heightened visibility in 2016.     

Conformational chemistry of surface-attached calmodulin detected by acoustic shear wave propagation

A thickness shear-mode acoustic wave device, operated in a flow-through format, was used to detect the binding of ions or peptides to surface-attached calmodulin. On-line surface attachment of the protein was achieved by immobilisation of the biotinylated molecule via a neutravidin-biotin linkage onto the surface of the gold electrode of the detector. The interaction between calmodulin, and calcium and magnesium ions induced an increase in resonant frequency and a decrease in motional resistance, which were reversible on washing with buffer. Interestingly, the changes in resonant frequency and motional resistance induced by the binding were opposite to the normal operation of the detector. The response was interpreted as a decrease in surface coupling (partial slip at the liquid/solid interface) instigated by exposure of hydrophobic domains on the protein, and an increase in the thickness, and hence effective wavelength, of the acoustic device, corresponding to an increase in the length of calmodulin by 1.5 A. This result is consistent with the literature value of 4 A. In addition, the interaction of the protein with peptide together with calcium ions was detected successfully, despite the relatively low molecular mass of the 2-kDa peptide. These results confirm the potential of acoustic wave physics for the detection of changes in the conformational chemistry of monolayer of biochemical macromolecules at the solid/liquid interface.     

Improvement of the fungal enzyme pyranose 2-oxidase using protein engineering

Native pyranose 2-oxidase (P2Ox) was purified from Peniophora sp. and characterized. To improve its catalytic efficiencies and stabilities by protein engineering, we cloned and expressed the P2Ox gene in Escherichia coli and received active, fully flavinylated recombinant P2OxA. Selenomethionine-labeled P2OxA was used for X-ray analysis and the resulting crystal structure enabled the rational design using variant P2OxA1 with the substitution E542K as template. Besides increased thermal and pH stabilities this variant showed improved catalytic efficiencies (k(cat)/K(m)) for the main substrates. A new variant, P2OxA2H, with an additional substitution T158A and a C-terminal His(6)-tag exhibited significantly decreased apparent K(m) values for D-glucose (0.47 mM), l-sorbose (1.79 mM), and D-xylose (1.35 mM). Compared to native P2Ox, the catalytic efficiencies were substantially improved for D-glucose (230-fold), L-sorbose (874-fold), and D-xylose (1751-fold). This P2Ox variant was used for the bioconversion of L-sorbose under O(2)-saturation in a molar scale. The structure-activity relationships of the amino acid substitutions were analyzed by modelling of the mutated P2Ox structures. Molecular docking calculations of various carbohydrates into the crystal structure of P2OxA and the analysis of the protein-ligand interactions in the docked complexes enabled us to explain the substrate specificity of the enzyme by a conserved hydrogen bond pattern which is formed between the protein and all substrates.     

Cloning, characterization, and engineering of fungal L-arabinitol dehydrogenases

L-Arabinitol 4-dehydrogenase (LAD) catalyzes the conversion of L-arabinitol to L-xylulose with concomitant NAD⁺ reduction in fungal L-arabinose catabolism. It is an important enzyme in the development of recombinant organisms that convert L-arabinose to fuels and chemicals. Here, we report the cloning, characterization, and engineering of four fungal LADs from Penicillium chrysogenum, Pichia guilliermondii, Aspergillus niger, and Trichoderma longibrachiatum, respectively. The LAD from P. guilliermondii was inactive, while the other three LADs were NAD⁺-dependent and showed high catalytic activities, with P. chrysogenum LAD being the most active. T. longibrachiatum LAD was the most thermally stable and showed the maximum activity in the temperature range of 55–65°C with the other LADs showed the maximum activity in the temperature range of 40–50°C. These LADs were active from pH 7 to 11 with an optimal pH of 9.4. Site-directed mutagenesis was used to alter the cofactor specificity of these LADs. In a T. longibrachiatum LAD mutant, the cofactor preference toward NADP⁺ was increased by 2.5 × 10⁴-fold, whereas the cofactor preference toward NADP⁺ of the P. chrysogenum and A. niger LAD mutants was also drastically improved, albeit at the expense of significantly reduced catalytic efficiencies. The wild-type LADs and their mutants with altered cofactor specificity could be used to investigate the functionality of the fungal L-arabinose pathways in the development of recombinant organisms for efficient microbial L-arabinose utilization.     

Chemical biotechnology for the specific oxyfunctionalization of hydrocarbons on a technical scale

Oxygenases catalyze, among other interesting reactions, highly selective hydrocarbon oxyfunctionalizations, which are important in industrial organic synthesis but difficult to achieve by chemical means. Many enzymatic oxygenations have been described, but few of these have been scaled up to industrial scales, due to the complexity of oxygenase based biocatalysts and demanding process implementation. We have combined recombinant whole-cell catalysis in a two-liquid phase system with fed-batch cultivation in an optimized medium and developed an industrially feasible process for the kinetically controlled and complex multistep oxidation of pseudocumene to 3,4-dimethylbenzaldehyde using the xylene monooxygenase of Pseudomonas putida mt-2 in Escherichia coli. Successful scale up to 30 L working volume using downscaled industrial equipment allowed a productivity of 31 g L(-1) d(-1) and a product concentration of 37 g L(-1). These performance characteristics meet present industry requirements. Product purification resulted in the recovery of 469 g of 3,4-dimethyl- benzaldehyde at a purity of 97% and an overall yield of 65%. This process illustrates the general feasibility of industrial biocatalytic oxyfunctionalization.     

Absence of unspecific innate immune cell activation by GATA-3-specific DNAzymes

DNAzymes of the 10-23 family represent an important class of antisense molecules with implications for therapeutic treatment of diseases. These molecules are single-stranded oligodeoxynucleotides combining the high specificity of oligonucleotide base pairing with an inherent RNA-cleaving enzymatic activity. However, like other oligonucleotide-based molecules these substances might exert so-called off-target effects, which have not been investigated so far for this molecule class. Therefore, the present study investigates putative off-target effects of DNAzymes on innate immune mechanisms using GATA-3-specific DNAzymes that have recently been developed as novel therapeutic approach for the treatment of allergic diseases including allergic asthma. The conserved catalytic domain of 10-23 DNAzymes contains a CpG motif that may stimulate innate immune cells via Toll-like receptor 9 (TLR-9). Therefore, potential TLR-9-mediated as well as TLR-9 independent cell activation was investigated using TLR-9-transfected HEK293 cells, macrophage cell lines and primary innate immune cells. Furthermore, putative effects of GATA-3-specific DNAzymes on the activation of neutrophil granulocytes and degranulation of mast cells/basophils were analyzed. In summary, no innate immune cell-stimulating activities of the tested DNAzymes were observed in any of the systems. Consequently, use of GATA-3-specific DNAzymes may represent a novel and highly specific approach for the treatment of allergic diseases.     

Epoxidation of linear,branched and cyclic alkenes catalyzed by unspecific peroxygenase

Unspecific peroxygenases (EC 1.11.2.1) represent a group of secreted heme-thiolate proteins that are capable of catalyzing the mono-oxygenation of diverse organic compounds, using only H₂O₂ as a co-substrate. Here we show that the peroxygenase secreted by the fungus Agrocybe aegerita catalyzed the oxidation of 20 different alkenes. Five branched alkenes, among them 2,3-dimethyl-2-butene and cis-2-butene, as well as propene and butadiene were epoxidized with complete regioselectivity. Longer linear alkenes with a terminal double bond (e.g. 1-octene) and cyclic alkenes (e.g. cyclohexene) were converted into the corresponding epoxides and allylic hydroxylation products; oxidation of the cyclic monoterpene limonene yielded three oxygenation products (two epoxides and an alcohol). In the case of 1-alkenes, the conversion occurred with moderate stereoselectivity, in which the preponderance for the (S)-enantiomer reached up to 72% ee for the epoxide product. The apparent Michaelis–Menten constant (K_m) for the epoxidation of the model substrate 2-methyl-2-butene was 5 mM, the turnover number (k_cat) 1.3 × 10³ s^?1 and the calculated catalytic efficiency, k_cat/K_m, was 2.5 × 10⁵ M^?1 s^?1. As epoxides represent chemical building blocks of high relevance, new enzymatic epoxidation pathways are of interest to complement existing chemical and biotechnological approaches. Stable and versatile peroxygenases as that of A. aegerita may form a promising biocatalytic platform for the development of such enzyme-based syntheses.     

Metabolic engineering of geranic acid in maize to achieve fungal resistance is compromised by novel glycosylation patterns

Many terpenoids are known to have antifungal properties and overexpression of these compounds in crops is a potential tool in disease control. In this study, 15 different mono- and sesquiterpenoids were tested in vitro against two major pathogenic fungi of maize (Zea mays), Colletotrichum graminicola and Fusarium graminearum. Among all tested terpenoids, geranic acid showed very strong inhibitory activity against both fungi (MIC<46 μM). To evaluate the possibility of enhancing fungal resistance in maize by overexpressing geranic acid, we generated transgenic plants with the geraniol synthase gene cloned from Lippia dulcis under the control of a ubiquitin promoter. The volatile and non-volatile metabolite profiles of leaves from transgenic and control lines were compared. The headspaces collected from intact seedlings of transgenic and control plants were not significantly different, although detached leaves of transgenic plants emitted 5-fold more geranyl acetate compared to control plants. Non-targeted LC-MS profiling and LC-MS-MS identification of extracts from maize leaves revealed that the major significantly different non-volatile compounds were 2 geranic acid derivatives, a geraniol dihexose and 4 different types of hydroxyl-geranic acid-hexoses. A geranic acid glycoside was the most abundant, and identified by NMR as geranoyl-6-O-malonyl-β-d-glucopyranoside with an average concentration of 45μM. Fungal bioassays with C. graminicola and F. graminearum did not reveal an effect of these changes in secondary metabolite composition on plant resistance to either fungus. The results demonstrate that metabolic engineering of geraniol into geranic acid can rely on the existing default pathway, but branching glycosylation pathways must be controlled to achieve accumulation of the aglycones.     

A new potent inhibitor of fungal melanin biosynthesis identified through combinatorial chemistry.

A new fungicide lead has been identified by in vitro screening of a focused combinatorial library. Amides (768) were synthesized in pools of four and assayed as inhibitors of scytalone dehydratase. Deconvolution of one of the most active pools led to the discovery of a potent inhibitor of the enzyme 3b (K(i) = 26 pM), which has fungicidal properties.     

Oxidative chemistry of 2-nitro and 4-nitroestradiol: Dichotomous behavior of radical intermediates and novel potential routes for oxyfunctionalization and B-ring fission of steroidal scaffolds

Oxidation of 4-nitro-17beta-estradiol (1) with the peroxidase/H(2)O(2) system gave the symmetric C(2)-linked dimer (3) through phenoxy radical coupling. Similar oxidation of 2-nitro-17beta-estradiol (2), in which the nitro group is coplanar with the aromatic ring, yielded 9alpha- and 9beta-hydroxy-2-nitro-17beta-estradiol (4a,b), (17beta)-2-nitroestra-1(10),2,4,9(11)-tetraene-3,17-diol (5), and (12alpha,17beta)-2-nitroestra-1(10),2,4,9(11)-tetraene-3,12,17-triol (6). With higher concentrations of H(2)O(2), the novel secoestra-1(10),2,4-trien-9-one derivative 7 was obtained from 2. Theoretical calculations suggested that the peculiar behavior of 2 may be due to the generation of a relatively stable radical intermediate at C(9), which would then be converted to the reactive quinone methide 8. The chemistry described in this paper appears to be an intriguing example of control of the site of substitution over evolution of phenoxy radicals, and opens new vistas toward selective oxyfunctionalization of the estrane skeleton.