Atomistic modelling and NMR studies reveal that gallium can target the ferric PQS uptake system in P. aeruginosa biofilms.

Intravenous gallium nitrate therapy is a novel therapeutic strategy deployed to combat chronic Pseudomonas aeruginosa biofilm infections in the lungs of cystic fibrosis (CF) patients by interfering with iron (Fe3+) uptake. The therapy is a source of Ga3+, which competes with Fe3+ for siderophore binding, subsequently disrupting iron metabolism and inhibiting biofilm proliferation in vivo. It was recently demonstrated that the Pseudomonas quinolone signal (PQS) can chelate Fe3+ to assist in bacterial iron uptake. However, it is unknown whether exogenous gallium also targets [Fe(PQS)3] uptake, which, in turn, would extend the mechanism of gallium therapy beyond siderophore competition, potentially supporting use of the therapy against P. aeruginosa mutants deficient in siderophore uptake proteins. To that end, the thermodynamic feasibility of iron-for-gallium cation exchange into [Fe(PQS)3] was evaluated using quantum chemical density functional theory (DFT) modelling and verified experimentally using 1H nuclear magnetic resonance (NMR). We demonstrate here that Ga3+ can strongly bind to three PQS molecules and, furthermore, displace and substitute Fe3+ from the native chelate pocket within PQS complexes, through a Trojan horse mechanism, retaining the key structural features present within the native ferric complex. As such, [Fe(PQS)3] complexes, in addition to ferric-siderophore complexes, represent another target for gallium therapy.

A Reductive Aminase Switches to Imine Reductase Mode for a Bulky Amine Substrate.

Imine reductases (IREDs) catalyze the asymmetric reduction of cyclic imines, but also in some cases the coupling of ketones and amines to form secondary amine products in an enzyme-catalyzed reductive amination (RedAm) reaction. Enzymatic RedAm reactions have typically used small hydrophobic amines, but many interesting pharmaceutical targets require that larger amines be used in these coupling reactions. Following the identification of IR77 from Ensifer adhaerens as a promising biocatalyst for the reductive amination of cyclohexanone with pyrrolidine, we have characterized the ability of this enzyme to catalyze couplings with larger bicyclic amines such as isoindoline and octahydrocyclopenta(c)pyrrole. By comparing the activity of IR77 with reductions using sodium cyanoborohydride in water, it was shown that, while the coupling of cyclohexanone and pyrrolidine involved at least some element of reductive amination, the amination with the larger amines likely occurred ex situ, with the imine recruited from solution for enzyme reduction. The structure of IR77 was determined, and using this as a basis, structure-guided mutagenesis, coupled with point mutations selecting improving amino acid sites suggested by other groups, permitted the identification of a mutant A208N with improved activity for amine product formation. Improvements in conversion were attributed to greater enzyme stability as revealed by X-ray crystallography and nano differential scanning fluorimetry. The mutant IR77-A208N was applied to the preparative scale amination of cyclohexanone at 50 mM concentration, with 1.2 equiv of three larger amines, in isolated yields of up to 93%.

Polyphosphinoborane Block Copolymer Synthesis Using Catalytic Reversible Chain-Transfer Dehydropolymerization.

An amphiphilic block copolymer of polyphosphinoborane has been prepared by a mechanism-led strategy of the sequential catalytic dehydropolymerization of precursor monomers, H3 B ⋅ PRH2 (R=Ph, n-hexyl), using the simple pre-catalyst [Rh(Ph2 PCH2 CH2 PPh2 )2 ]Cl. Speciation, mechanism and polymer chain growth studies support a step-growth process where reversible chain transfer occurs, i.e. H3 B ⋅ PRH2 /oligomer/polymer can all coordinate with, and be activated by, the catalyst. Block copolymer [H2 BPPhH]110 -b-[H2 BP(n-hexyl)H]11 can be synthesized and self-assembles in solution to form either rod-like micelles or vesicles depending on solvent polarity.

Ortho-aryl substituted DPEphos ligands: rhodium complexes featuring C-H anagostic interactions and B-H agostic bonds.

The synthesis of new Schrock-Osborn Rh(i) pre-catalysts with ortho-substituted DPEphos ligands, [Rh(DPEphos-R)(NBD)][BArF 4] [R = Me, OMe, iPr; ArF = 3,5-(CF3)2C6H3], is described. Along with the previously reported R = H variant, variable temperature 1H NMR spectroscopic and single-crystal X-ray diffraction studies show that these all have axial (C-H)⋯Rh anagostic interactions relative to the d8 pseudo square planar metal centres, that also result in corresponding downfield chemical shifts. Analysis by NBO, QTAIM and NCI methods shows these to be only very weak C-H⋯Rh bonding interactions, the magnitudes of which do not correlate with the observed chemical shifts. Instead, as informed by Scherer's approach, it is the topological positioning of the C-H bond with regard to the metal centre that is important. For [Rh(DPEphos-iPr)(NBD)][BArF 4] addition of H2 results in a Rh(iii) iPr-C-H activated product, [Rh(κ3,σ-P,O,P-DPEphos-iPr')(H)][BArF 4]. This undergoes H/D exchange with D2 at the iPr groups, reacts with CO or NBD to return Rh(i) products, and reaction with H3B·NMe3/tert-butylethene results in a dehydrogenative borylation to form a complex that shows both a non-classical B-H⋯Rh 3c-2e agostic bond and a C-H⋯Rh anagostic interaction at the same metal centre.

Fragment-derived modulators of an industrial ß-glucosidase.

A fragment screen of a library of 560 commercially available fragments using a kinetic assay identified a small molecule that increased the activity of the fungal glycoside hydrolase TrBgl2. An analogue by catalogue approach and detailed kinetic analysis identified improved compounds that behaved as nonessential activators with up to a 2-fold increase in maximum activation. The compounds did not activate the related bacterial glycoside hydrolase CcBglA demonstrating specificity. Interestingly, an analogue of the initial fragment inhibits both TrBgl2 and CcBglA, apparently through a mixed-model mechanism. Although it was not possible to determine crystal structures of activator binding to 55 kDa TrBgl2, solution NMR experiments demonstrated a specific binding site for the activator. A partial assignment of the NMR spectrum gave the identity of the amino acids at this site, allowing a model for TrBgl2 activation to be built. The activator binds at the entrance of the substrate-binding site, generating a productive conformation for the enzyme-substrate complex.

Correction to: 1H, 13C, 15N backbone and IVL methyl group resonance assignment of the fungal ß-glucosidase from Trichoderma reesei.

In the original publication of the article, the name of one of the authors is incorrect. The author's name is Eiso AB, but was modified to A. B. Eiso. The correct name is given in this Correction.

Profiling Substrate Promiscuity of Wild-Type Sugar Kinases for Multi-fluorinated Monosaccharides.

Fluorinated sugar-1-phosphates are of emerging importance as intermediates in the chemical and biocatalytic synthesis of modified oligosaccharides, as well as probes for chemical biology. Here we present a systematic study of the activity of a wide range of anomeric sugar kinases (galacto- and N-acetylhexosamine kinases) against a panel of fluorinated monosaccharides, leading to the first examples of polyfluorinated substrates accepted by this class of enzymes. We have discovered four new N-acetylhexosamine kinases with a different substrate scope, thus expanding the number of homologs available in this subclass of kinases. Lastly, we have solved the crystal structure of a galactokinase in complex with 2-deoxy-2-fluorogalactose, giving insight into changes in the active site that may account for the specificity of the enzyme toward certain substrate analogs.

1H, 13C, 15N backbone and IVL methyl group resonance assignment of the fungal ß-glucosidase from Trichoderma reesei.

ß-glucosidases have received considerable attention due to their essential role in bioethanol production from lignocellulosic biomass. ß-glucosidase can hydrolyse cellobiose in cellulose degradation and its low activity has been considered as one of the main limiting steps in the process. Large-scale conversions of cellulose therefore require high enzyme concentration which increases the cost. ß-glucosidases with improved activity and thermostability are therefore of great commercial interest. The fungus Trichoderma reseei expresses thermostable cellulolytic enzymes which have been widely studied as attractive targets for industrial applications. Genetically modified ß-glucosidases from Trichoderma reseei have been recently commercialised. We have developed an approach in which screening of low molecular weight molecules (fragments) identifies compounds that increase enzyme activity and are currently characterizing fragment-based activators of TrBgl2. A structural analysis of the 55 kDa apo form of TrBgl2 revealed a classical (α/ß)8-TIM barrel fold. In the present study we present a partial assignment of backbone chemical shifts, along with those of the Ile (I)-Val (V)-Leu (L) methyl groups of TrBgl2. These data will be used to characterize the interaction of TrBgl2 with the small molecule activators.

Conserved asymmetry underpins homodimerization of Dicer-associated double-stranded RNA-binding proteins.

Double-stranded RNA-binding domains (dsRBDs) are commonly found in modular proteins that interact with RNA. Two varieties of dsRBD exist: canonical Type A dsRBDs interact with dsRNA, while non-canonical Type B dsRBDs lack RNA-binding residues and instead interact with other proteins. In higher eukaryotes, the microRNA biogenesis enzyme Dicer forms a 1:1 association with a dsRNA-binding protein (dsRBP). Human Dicer associates with HIV TAR RNA-binding protein (TRBP) or protein activator of PKR (PACT), while Drosophila Dicer-1 associates with Loquacious (Loqs). In each case, the interaction involves a region of the protein that contains a Type B dsRBD. All three dsRBPs are reported to homodimerize, with the Dicer-binding region implicated in self-association. We report that these dsRBD homodimers display structural asymmetry and that this unusual self-association mechanism is conserved from flies to humans. We show that the core dsRBD is sufficient for homodimerization and that mutation of a conserved leucine residue abolishes self-association. We attribute differences in the self-association properties of Loqs, TRBP and PACT to divergence of the composition of the homodimerization interface. Modifications that make TRBP more like PACT enhance self-association. These data are examined in the context of miRNA biogenesis and the protein/protein interaction properties of Type B dsRBDs.

S6K2-mediated regulation of TRBP as a determinant of miRNA expression in human primary lymphatic endothelial cells.

MicroRNAs (miRNAs) are short non-coding RNAs that silence mRNAs. They are generated following transcription and cleavage by the DROSHA/DGCR8 and DICER/TRBP/PACT complexes. Although it is known that components of the miRNA biogenesis machinery can be phosphorylated, it remains poorly understood how these events become engaged during physiological cellular activation. We demonstrate that S6 kinases can phosphorylate the extended C-terminal domain of TRBP and interact with TRBP in situ in primary cells. TRBP serines 283/286 are essential for S6K-mediated TRBP phosphorylation, optimal expression of TRBP, and the S6K-TRBP interaction in human primary cells. We demonstrate the functional relevance of this interaction in primary human dermal lymphatic endothelial cells (HDLECs). Angiopoietin-1 (ANG1) can augment miRNA biogenesis in HDLECs through enhancing TRBP phosphorylation and expression in an S6K2-dependent manner. We propose that the S6K2/TRBP node controls miRNA biogenesis in HDLECs and provides a molecular link between the mTOR pathway and the miRNA biogenesis machinery.

Dissecting the roles of TRBP and PACT in double-stranded RNA recognition and processing of noncoding RNAs.

HIV TAR RNA-binding protein (TRBP) and Protein Activator of PKR (PACT) are double-stranded (ds) RNA-binding proteins that participate in both small regulatory RNA biogenesis and the response to viral dsRNA. Despite considerable progress toward understanding the structure-function relationship of TRBP and PACT, their specific roles in these seemingly distinct cellular pathways remain unclear. Both proteins are composed of three copies of the double-stranded RNA-binding domain, two of which interact with dsRNA, while the C-terminal copy mediates protein-protein interactions. PACT and TRBP are found in a complex with the endonuclease Dicer and facilitate processing of immature microRNAs. Their precise contribution to the Dicing step has not yet been defined: possibilities include precursor recruitment, rearrangement of dsRNA within the complex, loading the processed microRNA into the RNA-induced silencing complex, and distinguishing different classes of small dsRNA. TRBP and PACT also interact with the viral dsRNA sensors retinoic acid-inducible gene I (RIG-I) and double-stranded RNA-activated protein kinase (PKR). Current models suggest that PACT enables RIG-I to detect a wider range of viral dsRNAs, while TRBP and PACT exert opposing regulatory effects on PKR. Here, the evidence that implicates TRBP and PACT in regulatory RNA processing and viral dsRNA sensing is reviewed and discussed in the context of their molecular structure. The broader implications of a link between microRNA biogenesis and the innate antiviral response pathway are also considered.