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Abstract: In tryptophan biosynthesis, the reaction catalyzed by the enzyme indole-3-glycerol phosphate synthase (IGP synthase, IGPS) starts with a condensation step where the substrate’s carboxylated phenyl group makes a nucleophilic attack to form the pyrrole ring of the indole, followed by a decarboxylation which restores the aromaticity of the phenyl. IGPS from Pseudomonas aeruginosa has the highest turnover number of all characterized IGPS enzymes, providing an excellent model system to test the necessity of the decarboxylation step. This step was since the 1960s considered to be mechanistically essential based on studies of the IGPS:PRAI fusion protein from Escherichia coli. Here, we present the crystal structure of P. aeruginosa IGPS in complex with reduced CdRP, a non-reactive substrate analogue, and using a sensitive discontinuous assay, we demonstrate weak promiscuous activity on the decarboxylated substrate 1-(phenylamino)-1-deoxyribulose-5-phosphate (PAdRP) – with approximately 1000' lower rate of IGP formation than from the native substrate. We also show that E. coli IGPS, at even lower rate, can produce IGP from decarboxylated substrate. Our structure of P. aeruginosa IGPS has eight molecules in the asymmetric unit, out of which seven contain ligand, and one displays a previously unobserved conformation closer to the reactive state. One of the few non-conserved active-site residues, Phe201 in P. aeruginosa IGPS, is by mutagenesis demonstrated to be important for the higher turnover of this enzyme on both substrates. Our results demonstrate that, despite IGPS’s classification as a carboxylyase (i.e. decarboxylase), decarboxylation not a completely essential step in its catalysis.

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Abstract: Centrioles are key eukaryotic organelles responsible for the formation of cilia and flagella, and for organising the microtubule network and the mitotic spindle in animals. Centriole assembly requires oligomerisation of the essential protein spindle assembly abnormal 6 (SAS-6), which forms a structural scaffold templating the organisation of further organelle components. A dimerisation interaction between SAS-6 N-terminal ‘head’ domains was previously shown to be essential for protein oligomerisation in vitro and for function in centriole assembly. Here, we developed a pharmacophore model allowing us to assemble a library of low molecular weight ligands predicted to bind the SAS-6 head domain and inhibit protein oligomerisation. We demonstrate using nuclear magnetic resonance spectroscopy that a ligand from this family binds at the head domain dimerisation site of algae, nematode and human SAS-6 variants, but also that another ligand specifically recognises human SAS-6. Atomistic molecular dynamics simulations starting from SAS-6 head domain crystallographic structures, including that of the human head domain which we now resolve, suggest ligand specificity derives from favourable Van der Waals interactions with a hydrophobic cavity at the dimerisation site.

Open Access

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Abstract: Hypothesis: We define contact angles, h, during displacement of three fluid phases in a porous medium using energy balance, extending previous work on two-phase flow. We test if this theory can be applied to quantify the three contact angles and wettability order in pore-scale images of three-phase displacement. Theory: For three phases labelled 1, 2 and 3, and solid, s, using conservation of energy ignoring viscous dissipation ðDa1s cos h12  Da12  /j12 DS1 Þr12 1⁄4 ðDa3s cos h23 þ Da23  /j23 DS3 Þr23 þ Da13 r13 , where / is the porosity, r is the interfacial tension, a is the specific interfacial area, S is the saturation, and j is the fluid–fluid interfacial curvature. D represents the change during a displacement. The third contact angle, h13 can be found using the Bartell-Osterhof relationship. The energy balance is also extended to an arbitrary number of phases. Findings: X-ray imaging of porous media and the fluids within them, at pore-scale resolution, allows the difference terms in the energy balance equation to be measured. This enables wettability, the contact angles, to be determined for complex displacements, to characterize the behaviour, and for input into pore-scale models. Two synchrotron imaging datasets are used to illustrate the approach, comparing the flow of oil, water and gas in a water-wet and an altered-wettability limestone rock sample. We show that in the water-wet case, as expected, water (phase 1) is the most wetting phase, oil (phase 2) is inter- mediate wet, while gas (phase 3) is most non-wetting with effective contact angles of h12 % 48 and h13 % 44, while h23 1⁄4 0 since oil is always present in spreading layers. In contrast, for the altered-wettability case, oil is most wetting, gas is intermediate-wet, while water is most non-wetting with con- tact angles of h12 1⁄4 134 $ 10;h13 1⁄4 119 $ 10, and h23 1⁄4 66 $ 10.

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Abstract: The fundamental kinetics of the electrocatalytic sulfur reduction reaction (SRR), a complex 16-electron conversion process in lithium–sulfur batteries, is so far insufficiently explored. Here, by directly profiling the activation energies in the multistep SRR, we reveal that the initial reduction of sulfur to the soluble polysulfides is relatively easy owing to the low activation energy, whereas the subsequent conversion of the polysulfides into the insoluble Li2S2/Li2S has a much higher activation energy, contributing to the accumulation of polysulfides and exacerbating the polysulfide shuttling effect. We use heteroatom-doped graphene as a model system to explore electrocatalytic SRR. We show that nitrogen and sulfur dual-doped graphene considerably reduces the activation energy to improve SRR kinetics. Density functional calculations confirm that the doping tunes the p-band centre of the active carbons for an optimal adsorption strength of intermediates and electroactivity. This study establishes electrocatalysis as a promising pathway to tackle the fundamental challenges facing lithium–sulfur batteries.

Open Access

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Abstract: Reductive dehalogenases are responsible for the reductive cleavage of carbon-halogen bonds during organohalide respiration. A variety of mechanisms have been proposed for these cobalamin and [4Fe-4S] containing enzymes, including organocobalt, radical, or cobalt-halide adduct based catalysis. The latter was proposed for the oxygen-tolerant Nitratireductor pacificus pht-3B catabolic reductive dehalogenase (NpRdhA). Here, we present the first substrate bound NpRdhA crystal structures, confirming a direct cobalt–halogen interaction is established and providing a rationale for substrate preference. Product formation is observed in crystallo due to X-ray photoreduction. Protein engineering enables rational alteration of substrate preference, providing a future blue print for the application of this and related enzymes in bioremediation.