Peri active site catalysis of proline isomerisation is the molecular basis of allomorphy in β-phosphoglucomutase

Metabolic regulation occurs through precise control of enzyme activity. Allomorphy is a post-translational fine control mechanism where the catalytic rate is governed by a conformational switch that shifts the enzyme population between forms with different activities. β-Phosphoglucomutase (βPGM) uses allomorphy in the catalysis of isomerisation of β-glucose 1-phosphate to glucose 6-phosphate via β-glucose 1,6-bisphosphate. Herein, we describe structural and biophysical approaches to reveal its allomorphic regulatory mechanism. Binding of the full allomorphic activator β-glucose 1,6-bisphosphate stimulates enzyme closure, progressing through NAC I and NAC III conformers. Prior to phosphoryl transfer, loops positioned on the cap and core domains are brought into close proximity, modulating the environment of a key proline residue. Hence accelerated isomerisation, likely via a twisted anti/C4-endo transition state, leads to the rapid predominance of active cis-P βPGM. In contrast, binding of the partial allomorphic activator fructose 1,6-bisphosphate arrests βPGM at a NAC I conformation and phosphoryl transfer to both cis-P βPGM and trans-P βPGM occurs slowly. Thus, allomorphy allows a rapid response to changes in food supply while not otherwise impacting substantially on levels of important metabolites

Engineering of B800 bacteriochlorophyll binding site specificity in the Rhodobacter sphaeroides LH2 antenna.

The light-harvesting 2 complex (LH2) of the purple phototrophic bacterium Rhodobacter sphaeroides is a highly efficient, light-harvesting antenna that allows growth under a wide-range of light intensities. In order to expand the spectral range of this antenna complex, we first used a series of competition assays to measure the capacity of the non-native pigments 3-acetyl chlorophyll (Chl) a, Chl d, Chl f or bacteriochlorophyll (BChl) b to replace native BChl a in the B800 binding site of LH2. We then adjusted the B800 site and systematically assessed the binding of non-native pigments. We find that Arg-10 of the LH2 β polypeptide plays a crucial role in binding specificity, by providing a hydrogen-bond to the 3-acetyl group of native and non-native pigments. Reconstituted LH2 complexes harbouring the series of (B)Chls were examined by transient absorption and steady-state fluorescence spectroscopies. Although slowed 10-fold to ~6 ps, energy transfer from Chl a to B850 BChl a remained highly efficient. We measured faster energy-transfer time constants for Chl d (3.5 ps) and Chl f (2.7 ps), which have red-shifted absorption maxima compared to Chl a. BChl b, red-shifted from the native BChl a, gave extremely rapid (≤0.1 ps) transfer. These results show that modified LH2 complexes, combined with engineered (B)Chl biosynthesis pathways in vivo, have potential for retaining high efficiency whilst acquiring increased spectral range

On the mechanisms governing gas penetration into a tokamak plasma during a massive gas injection

A new 1D radial fluid code, IMAGINE, is used to simulate the penetration of gas into a tokamak plasma during a massive gas injection (MGI). The main result is that the gas is in general strongly braked as it reaches the plasma, due to mechanisms related to charge exchange and (to a smaller extent) recombination. As a result, only a fraction of the gas penetrates into the plasma. Also, a shock wave is created in the gas which propagates away from the plasma, braking and compressing the incoming gas. Simulation results are quantitatively consistent, at least in terms of orders of magnitude, with experimental data for a D 2 MGI into a JET Ohmic plasma. Simulations of MGI into the background plasma surrounding a runaway electron beam show that if the background electron density is too high, the gas may not penetrate, suggesting a possible explanation for the recent results of Reux et al in JET (2015 Nucl. Fusion 55 093013)