Arginine Kinase Activates Arginine for Phosphorylation by Pyramidalization and Polarization.

Arginine phosphorylation plays numerous roles throughout biology. Arginine kinase (AK) catalyzes the delivery of an anionic phosphoryl group (PO3-) from ATP to a planar, trigonal nitrogen in a guanidinium cation. Density functional theory (DFT) calculations have yielded a model of the transition state (TS) for the AK-catalyzed reaction. They reveal a network of over 50 hydrogen bonds that delivers unprecedented pyramidalization and out-of-plane polarization of the arginine guanidinium nitrogen (Nη2) and aligns the electron density on Nη2 with the scissile P-O bond, leading to in-line phosphoryl transfer via an associative mechanism. In the reverse reaction, the hydrogen-bonding network enforces the conformational distortion of a bound phosphoarginine substrate to increase the basicity of Nη2. This enables Nη2 protonation, which triggers PO3- migration to generate ATP. This polarization-pyramidalization of nitrogen in the arginine side chain is likely a general phenomenon that is exploited by many classes of enzymes mediating the post-translational modification of arginine.

Molecular basis of specificity and deamidation of eIF4A by Burkholderia Lethal Factor 1.

Burkholderia pseudomallei lethal factor 1 (BLF1) exhibits site-specific glutamine deamidase activity against the eukaryotic RNA helicase, eIF4A, thereby blocking mammalian protein synthesis. The structure of a complex between BLF1 C94S and human eIF4A shows that the toxin binds in the cleft between the two RecA-like eIF4A domains forming interactions with residues from both and with the scissile amide of the target glutamine, Gln339, adjacent to the toxin active site. The RecA-like domains adopt a radically twisted orientation compared to other eIF4A structures and the nature and position of conserved residues suggests this may represent a conformation associated with RNA binding. Comparison of the catalytic site of BLF1 with other deamidases and cysteine proteases reveals that they fall into two classes, related by pseudosymmetry, that present either the re or si faces of the target amide/peptide to the nucleophilic sulfur, highlighting constraints in the convergent evolution of their Cys-His active sites.

Global land cover trajectories and transitions.

Global land cover (LC) changes threaten sustainability and yet we lack a comprehensive understanding of the gains and losses of LC types, including the magnitudes, locations and timings of transitions. We used a novel, fine-resolution and temporally consistent satellite-derived dataset covering the entire Earth annually from 1992 to 2018 to quantify LC changes across a range of scales. At global and continental scales, the observed trajectories of change for most LC types were fairly smooth and consistent in direction through time. We show these observed trajectories in the context of error margins produced by extrapolating previously published accuracy metrics associated with the LC dataset. For many LC classes the observed changes were found to be within the error margins. However, an important exception was the increase in urban land, which was consistently larger than the error margins, and for which the LC transition was unidirectional. An advantage of analysing the global, fine spatial resolution LC time-series dataset is the ability to identify where and when LC changes have taken place on the Earth. We present LC change maps and trajectories that identify locations with high dynamism, and which pose significant sustainability challenges. We focused on forest loss and urban growth at the national scale, identifying the top 10 countries with the largest percentages of forest loss and urban growth globally. Crucially, we found that most of these 'worst-case' countries have stabilized their forest losses, although urban expansion was monotonic in all cases. These findings provide crucial information to support progress towards the UN's SDGs.

Octahedral Trifluoromagnesate, an Anomalous Metal Fluoride Species, Stabilizes the Transition State in a Biological Motor.

Isoelectronic metal fluoride transition state analogue (TSA) complexes, MgF3 - and AlF4 -, have proven to be immensely useful in understanding mechanisms of biological motors utilizing phosphoryl transfer. Here we report a previously unobserved octahedral TSA complex, MgF3(H2O)-, in a 1.5 Å resolution Zika virus NS3 helicase crystal structure. 19F NMR provided independent validation and also the direct observation of conformational tightening resulting from ssRNA binding in solution. The TSA stabilizes the two conformations of motif V of the helicase that link ATP hydrolysis with mechanical work. DFT analysis further validated the MgF3(H2O)- species, indicating the significance of this TSA for studies of biological motors.

Multiscale computation delivers organophosphorus reactivity and stereoselectivity to immunoglobulin scavengers.

Quantum mechanics/molecular mechanics (QM/MM) maturation of an immunoglobulin (Ig) powered by supercomputation delivers novel functionality to this catalytic template and facilitates artificial evolution of biocatalysts. We here employ density functional theory-based (DFT-b) tight binding and funnel metadynamics to advance our earlier QM/MM maturation of A17 Ig-paraoxonase (WTIgP) as a reactibody for organophosphorus toxins. It enables regulation of biocatalytic activity for tyrosine nucleophilic attack on phosphorus. The single amino acid substitution l-Leu47Lys results in 340-fold enhanced reactivity for paraoxon. The computed ground-state complex shows substrate-induced ionization of the nucleophilic l-Tyr37, now H-bonded to l-Lys47, resulting from repositioning of l-Lys47. Multiple antibody structural homologs, selected by phenylphosphonate covalent capture, show contrasting enantioselectivities for a P-chiral phenylphosphonate toxin. That is defined by crystallographic analysis of phenylphosphonylated reaction products for antibodies A5 and WTIgP. DFT-b analysis using QM regions based on these structures identifies transition states for the favored and disfavored reactions with surprising results. This stereoselection analysis is extended by funnel metadynamics to a range of WTIgP variants whose predicted stereoselectivity is endorsed by experimental analysis. The algorithms used here offer prospects for tailored design of highly evolved, genetically encoded organophosphorus scavengers and for broader functionalities of members of the Ig superfamily, including cell surface-exposed receptors.

Genomic and Metabolomic Polymorphism among Experimentally Selected Paromomycin-Resistant Leishmania donovani Strains.

Understanding the mechanism(s) underpinning drug resistance could lead to novel treatments to reverse the increased tolerance of a pathogen. In this study, paromomycin (PMM) resistance (PMMr) was induced in three Nepalese clinical strains of Leishmania donovani with different inherent susceptibilities to antimony (Sb) drugs by stepwise exposure of promastigotes to PMM. Exposure to PMM resulted in the production of mixed populations of parasites, even though a single cloned population was used at the start of selection. PMM 50% inhibitory concentration (IC50) values for PMMr parasites varied between 104 and 481 µM at the promastigote stage and 32 and 195 µM at the intracellular amastigote stage. PMM resistance was associated with increased resistance to nitric oxide at the amastigote stage but not the promastigote stage (P < 0.05). This effect was most marked in the Sb-resistant (Sbr) PMMr clone, in which PMM resistance was associated with a significant upregulation of glutathione compared to that in its wild type (P < 0.05), although there was no change in the regulation of trypanothione (detected in its oxidized form). Interestingly, PMMr strains showed an increase in either the keto acid derivative of isoleucine (Sb intermediate PMMr) or the 2-hydroxy acids derived from arginine and tyrosine (Sb susceptible PMMr and Sbr PMMr). These results are consistent with the recent finding that the upregulation of the branched-chain amino acid aminotransferase and d-lactate dehydrogenase is linked to PMMr In addition, we found that PMMr is associated with a significant increase in aneuploidy during PMM selection in all the strains, which could allow the rapid selection of genetic changes that confer a survival advantage.

Thermokinetic profile of NDM-1 and its inhibition by small carboxylic acids.

The New Delhi metallo-ß-lactamase (NDM-1) is an important clinical target for antimicrobial research, but there are insufficient clinically useful inhibitors and the details of NDM-1 enzyme catalysis remain unclear. The aim of this work is to provide a thermodynamic profile of NDM-1 catalysed hydrolysis of ß-lactams using an isothermal titration calorimetry (ITC) approach and to apply this new method to the identification of new low-molecular-weight dicarboxylic acid inhibitors. The results reveal that hydrolysis of penicillin G and imipenem by NDM-1 share the same thermodynamic features with a significant intrinsic enthalpy change and the release of one proton into solution, while NDM-1 hydrolysis of cefazolin exhibits a different mechanism with a smaller enthalpy change and the release of two protons. The inhibitory constants of four carboxylic acids are found to be in the micromolar range. The compounds pyridine-2,6-dicarboxylic acid and thiazolidine-2,4-dicarboxylic acid show the best inhibitory potency and are confirmed to inhibit NDM-1 using a clinical strain of Escherichia coli The pyridine compound is further shown to restore the susceptibility of this E. coli strain to imipenem, at an inhibitor concentration of 400 µM, while the thiazoline compound also shows a synergistic effect with imipenem. These results provide valuable information to enrich current understanding on the catalytic mechanism of NDM-1 and to aid the future optimisation of ß-lactamase inhibitors based on these scaffolds to tackle the problem of antibiotic resistance.

Quantifying the exposure of humans and the environment to oil pollution in the Niger Delta using advanced geostatistical techniques.

The Niger Delta is one of the largest oil producing regions of the world. Large numbers and volumes of oil spills have been reported in this region. What has not been quantified is the putative exposure of humans and/or the environment to this hydrocarbon pollution. In this novel study, advanced geostatistical techniques were applied to an extensive database of oil spill incidents from 2007 to 2015. The aims were to (i) identify and analyse spill hotspots along the oil pipeline network and (ii) estimate the exposure of the hydrocarbon pollution to the human population and the environment within the Niger Delta. Over the study period almost 90millionlitres of oil were released. Approximately 29% of the human population living in proximity to the pipeline network has been potentially exposed to oil contamination, of which 565,000 people live within high or very high spill intensity sectors. Over 1000km2 of land has been contaminated by oil pollution, with broadleaved forest, mangroves and agricultural land the most heavily impacted land cover types. Proximity to the coast, roads and cities are the strongest spatial factors contributing to spill occurrence, which largely determine the accessibility of sites for pipeline sabotage and oil theft. Overall, the findings demonstrate the high levels of environmental and human exposure to hydrocarbon pollutants in the Niger Delta. These results provide evidence with which to spatially target interventions to reduce future spill incidents and mitigate the impacts of previous spills on human communities and ecosystem health.

Evolution of catalytic centers of antibodies by virtual screening of broad repertoire of mutants using supercomputer.

It is proposed to perform quantum mechanical/molecular dynamics calculations of chemical reactions that are planned to be catalyzed by antibodies and then conduct a virtual screening of the library of potential antibody mutants to select an optimal biocatalyst. We tested the effectiveness of this approach by the example of hydrolysis of organophosphorus toxicant paraoxon using kinetic approaches and X-ray analysis of the antibody biocatalyst designed de novo.

Assessing the Influence of Mutation on GTPase Transition States by Using X-ray Crystallography, 19 F†NMR, and DFT Approaches.

We report X-ray crystallographic and 19 F NMR studies of the G-protein RhoA complexed with MgF3- , GDP, and RhoGAP, which has the mutation Arg85'Ala. When combined with DFT calculations, these data permit the identification of changes in transition state (TS) properties. The X-ray data show how Tyr34 maintains solvent exclusion and the core H-bond network in the active site by relocating to replace the missing Arg85' sidechain. The 19 F NMR data show deshielding effects that indicate the main function of Arg85' is electronic polarization of the transferring phosphoryl group, primarily mediated by H-bonding to O3G and thence to PG . DFT calculations identify electron-density redistribution and pinpoint why the TS for guanosine 5'-triphosphate (GTP) hydrolysis is higher in energy when RhoA is complexed with RhoGAPArg85'Ala relative to wild-type (WT) RhoGAP. This study demonstrates that 19 F NMR measurements, in combination with X-ray crystallography and DFT calculations, can reliably dissect the response of small GTPases to site-specific modifications.

Metal Fluorides: Tools for Structural and Computational Analysis of Phosphoryl Transfer Enzymes.

The phosphoryl group, PO3-, is the dynamic structural unit in the biological chemistry of phosphorus. Its transfer from a donor to an acceptor atom, with oxygen much more prevalent than nitrogen, carbon, or sulfur, is at the core of a great majority of enzyme-catalyzed reactions involving phosphate esters, anhydrides, amidates, and phosphorothioates. The serendipitous discovery that the phosphoryl group could be labeled by "nuclear mutation," by substitution of PO3- by MgF3- or AlF4-, has underpinned the application of metal fluoride (MF x ) complexes to mimic transition states for enzymatic phosphoryl transfer reactions, with sufficient stability for experimental analysis. Protein crystallography in the solid state and 19F NMR in solution have enabled direct observation of ternary and quaternary protein complexes embracing MF x transition state models with precision. These studies have underpinned a radically new mechanistic approach to enzyme catalysis for a huge range of phosphoryl transfer processes, as varied as kinases, phosphatases, phosphomutases, and phosphohydrolases. The results, without exception, have endorsed trigonal bipyramidal geometry (tbp) for concerted, "in-line" stereochemistry of phosphoryl transfer. QM computations have established the validity of tbp MF x complexes as reliable models for true transition states, delivering similar bond lengths, coordination to essential metal ions, and virtually identical hydrogen bond networks. The emergence of protein control of reactant orbital overlap between bond-forming species within enzyme transition states is a new challenging theme for wider exploration.

Metal Fluorides as Analogues for Studies on Phosphoryl Transfer Enzymes.

The 1994 structure of a transition-state analogue with AlF4- and GDP complexed to G1α, a small G protein, heralded a new field of research into the structure and mechanism of enzymes that manipulate the transfer of phosphoryl (PO3- ) groups. The number of enzyme structures in the PDB containing metal fluorides (MFx ) as ligands that imitate either a phosphoryl or a phosphate group was 357 at the end of 2016. They fall into three distinct geometrical classes: 1) Tetrahedral complexes based on BeF3- that mimic ground-state phosphates; 2) octahedral complexes, primarily based on AlF4- , which mimic "in-line" anionic transition states for phosphoryl transfer; and 3) trigonal bipyramidal complexes, represented by MgF3- and putative AlF30 moieties, which mimic the geometry of the transition state. The interpretation of these structures provides a deeper mechanistic understanding into the behavior and manipulation of phosphate monoesters in molecular biology. This Review provides a comprehensive overview of these structures, their uses, and their computational development.

Robotic QM/MM-driven maturation of antibody combining sites.

In vitro selection of antibodies from large repertoires of immunoglobulin (Ig) combining sites using combinatorial libraries is a powerful tool, with great potential for generating in vivo scavengers for toxins. However, addition of a maturation function is necessary to enable these selected antibodies to more closely mimic the full mammalian immune response. We approached this goal using quantum mechanics/molecular mechanics (QM/MM) calculations to achieve maturation in silico. We preselected A17, an Ig template, from a naïve library for its ability to disarm a toxic pesticide related to organophosphorus nerve agents. Virtual screening of 167,538 robotically generated mutants identified an optimum single point mutation, which experimentally boosted wild-type Ig scavenger performance by 170-fold. We validated the QM/MM predictions via kinetic analysis and crystal structures of mutant apo-A17 and covalently modified Ig, thereby identifying the displacement of one water molecule by an arginine as delivering this catalysis.

(19)F†NMR and DFT Analysis Reveal Structural and Electronic Transition State Features for RhoA-Catalyzed GTP Hydrolysis.

Molecular details for RhoA/GAP catalysis of the hydrolysis of GTP to GDP are poorly understood. We use (19)F NMR chemical shifts in the MgF3(-) transition state analogue (TSA) complex as a spectroscopic reporter to indicate electron distribution for the γ-PO3(-) oxygens in the corresponding TS, implying that oxygen coordinated to Mg has the greatest electron density. This was validated by QM calculations giving a picture of the electronic properties of the transition state (TS) for nucleophilic attack of water on the γ-PO3(-) group based on the structure of a RhoA/GAP-GDP-MgF3(-) TSA complex. The TS model displays a network of 20 hydrogen bonds, including the GAP Arg85' side chain, but neither phosphate torsional strain nor general base catalysis is evident. The nucleophilic water occupies a reactive location different from that in multiple ground state complexes, arising from reorientation of the Gln-63 carboxamide by Arg85' to preclude direct hydrogen bonding from water to the target γ-PO3(-) group.

In vitro selection of miltefosine resistance in promastigotes of Leishmania donovani from Nepal: genomic and metabolomic characterization.

In this study, we followed the genomic, lipidomic and metabolomic changes associated with the selection of miltefosine (MIL) resistance in two clinically derived Leishmania donovani strains with different inherent resistance to antimonial drugs (antimony sensitive strain Sb-S; and antimony resistant Sb-R). MIL-R was easily induced in both strains using the promastigote-stage, but a significant increase in MIL-R in the intracellular amastigote compared to the corresponding wild-type did not occur until promastigotes had adapted to 12.2 µM MIL. A variety of common and strain-specific genetic changes were discovered in MIL-adapted parasites, including deletions at the LdMT transporter gene, single-base mutations and changes in somy. The most obvious lipid changes in MIL-R promastigotes occurred to phosphatidylcholines and lysophosphatidylcholines and results indicate that the Kennedy pathway is involved in MIL resistance. The inherent Sb resistance of the parasite had an impact on the changes that occurred in MIL-R parasites, with more genetic changes occurring in Sb-R compared with Sb-S parasites. Initial interpretation of the changes identified in this study does not support synergies with Sb-R in the mechanisms of MIL resistance, though this requires an enhanced understanding of the parasite's biochemical pathways and how they are genetically regulated to be verified fully.

A novel expression cassette delivers efficient production of exclusively tetrameric human butyrylcholinesterase with improved pharmacokinetics for protection against organophosphate poisoning.

Butyrylcholinesterase is a stoichiometric bioscavenger against poisoning by organophosphorus pesticides and nerve agents. The low level of expression and extremely rapid clearance of monomeric recombinant human butyrylcholinesterase (rhBChE) from bloodstream (t½≈2 min) limits its pharmaceutical application. Recently (Ilyushin at al., PNAS, 2013) we described a long-acting polysialylated recombinant butyrylcholinesterase (rhBChE-CAO), stable in the bloodstream, that protects mice against 4.2 LD50 of VR. Here we report a set of modifications of the initial rhBChE expression vector to improve stability of the enzyme in the bloodstream and increase its production in CHO cells by introducing in the expression cassette: (i) the sequence of the natural human PRAD-peptide in frame with rhBChE gene via "self-processing" viral F2A peptide under control of an hEF/HTLV promoter, and (ii) previously predicted in silico MAR 1-68 and MAR X-29 sequences. This provides fully tetrameric rhBChE (4rhBChE) at 70 mg/l, that displays improved pharmacokinetics (t½ = 32 ± 1.2 h, MRT = 43 ± 2 h). 3D Fluorescent visualization and distribution of (125)I-labeled enzyme reveals similar low level 4rhBChE and rhBChE-CAO accumulation in muscle, fat, and brain. Administered 4rhBChE was mainly catabolized in the liver and breakdown products were excreted in kidney. Injection of 1.2 LD50 and 1.1 LD50 of paraoxon to BALB/c and knockout BChE-/- mice pre-treated with 4rhBChE (50 mg/kg) resulted in 100% and 78% survival, respectively, without perturbation of long-term behavior. In contrast, 100% mortality of non-pre-treated mice was observed. The high expression level of 4rhBChE in CHO cells permits consideration of this new expression system for manufacturing BChE as a biopharmaceutical.

α-Fluorophosphonates reveal how a phosphomutase conserves transition state conformation over hexose recognition in its two-step reaction.

ß-Phosphoglucomutase (ßPGM) catalyzes isomerization of ß-D-glucose 1-phosphate (ßG1P) into D-glucose 6-phosphate (G6P) via sequential phosphoryl transfer steps using a ß-D-glucose 1,6-bisphosphate (ßG16BP) intermediate. Synthetic fluoromethylenephosphonate and methylenephosphonate analogs of ßG1P deliver novel step 1 transition state analog (TSA) complexes for ßPGM, incorporating trifluoromagnesate and tetrafluoroaluminate surrogates of the phosphoryl group. Within an invariant protein conformation, the ß-D-glucopyranose ring in the ßG1P TSA complexes (step 1) is flipped over and shifted relative to the G6P TSA complexes (step 2). Its equatorial hydroxyl groups are hydrogen-bonded directly to the enzyme rather than indirectly via water molecules as in step 2. The (C)O-P bond orientation for binding the phosphate in the inert phosphate site differs by ∼ 30° between steps 1 and 2. By contrast, the orientations for the axial O-Mg-O alignment for the TSA of the phosphoryl group in the catalytic site differ by only ∼ 5°, and the atoms representing the five phosphorus-bonded oxygens in the two transition states (TSs) are virtually superimposable. The conformation of ßG16BP in step 1 does not fit into the same invariant active site for step 2 by simple positional interchange of the phosphates: the TS alignment is achieved by conformational change of the hexose rather than the protein.

Effects of individual and combined dietary weight loss and exercise interventions in postmenopausal women on adiponectin and leptin levels.

BACKGROUND: Excess body weight and a sedentary lifestyle are associated with the development of several diseases, including cardiovascular disease, diabetes and cancer in women. One proposed mechanism linking obesity to chronic diseases is an alteration in adipose-derived adiponectin and leptin levels. We investigated the effects of 12-month reduced calorie, weight loss and exercise interventions on adiponectin and leptin concentrations. METHODS: Overweight/obese postmenopausal women (n = 439) were randomized as follows: (i) a reduced calorie, weight-loss diet (diet; N = 118), (ii) moderate-to-vigorous intensity aerobic exercise (exercise; N = 117), (iii) a combination of a reduced calorie, weight-loss diet and moderate-to-vigorous intensity aerobic exercise (diet + exercise; N = 117), and (iv) control (N = 87). The reduced calorie diet had a 10% weight-loss goal. The exercise intervention consisted of 45 min of moderate-to-vigorous aerobic activity 5 days per week. Adiponectin and leptin levels were measured at baseline and after 12 months of intervention using a radioimmunoassay. RESULTS: Adiponectin increased by 9.5% in the diet group and 6.6% in the diet + exercise group (both P ≤ 0.0001 vs. control). Compared with controls, leptin decreased with all interventions (diet + exercise, -40.1%, P < 0.0001; diet, -27.1%, P < 0.0001; exercise, -12.7%, P = 0.005). The results were not influenced by the baseline body mass index (BMI). The degree of weight loss was inversely associated with concentrations of adiponectin (diet, P-trend = 0.0002; diet + exercise, P-trend = 0.0005) and directly associated with leptin (diet, P-trend < 0.0001; diet + exercise, P-trend < 0.0001). CONCLUSION: Weight loss through diet or diet + exercise increased adiponectin concentrations. Leptin concentrations decreased in all of the intervention groups, but the greatest reduction occurred with diet + exercise. Weight loss and exercise exerted some beneficial effects on chronic diseases via effects on adiponectin and leptin.

Chemical polysialylation of human recombinant butyrylcholinesterase delivers a long-acting bioscavenger for nerve agents in vivo.

The creation of effective bioscavengers as a pretreatment for exposure to nerve agents is a challenging medical objective. We report a recombinant method using chemical polysialylation to generate bioscavengers stable in the bloodstream. Development of a CHO-based expression system using genes encoding human butyrylcholinesterase and a proline-rich peptide under elongation factor promoter control resulted in self-assembling, active enzyme multimers. Polysialylation gives bioscavengers with enhanced pharmacokinetics which protect mice against 4.2 LD(50) of S-(2-(diethylamino)ethyl) O-isobutyl methanephosphonothioate without perturbation of long-term behavior.

Reflections on biocatalysis involving phosphorus.

Early studies on chemical synthesis of biological molecules can be seen to progress to preparation and biological evaluation of phosphonates as analogues of biological phosphates, with emphasis on their isosteric and isopolar character. Work with such mimics progressed into structural studies with a range of nucleotide-utilising enzymes. The arrival of metal fluorides as analogues of the phosphoryl group, PO(3)(-), for transition state (TS) analysis of enzyme reactions stimulated the symbiotic deployment of (19)F NMR and protein crystallography. Characteristics of enzyme transition state analogues are reviewed for a range of reactions. From the available MF(x) species, trifluoroberyllate gives tetrahedral mimics of ground states (GS) in which phosphate is linked to carboxylate and phosphate oxyanions. Tetrafluoroaluminate is widely employed as a TS mimic, but it necessarily imposes octahedral geometry on the assembled complexes, whereas phosphoryl transfer involves trigonal bipyramidal (tbp) geometry. Trifluoromagnesate (MgF(3)(-)) provides the near-ideal solution, delivering tbp geometry and correct anionic charge. Some of the forty reported tbp structures assigned as having AlF(3)(0) cores have been redefined as trifluoromagnesate complexes. Transition state analogues for a range of kinases, mutases, and phosphatases provide a detailed description of mechanism for phosphoryl group transfer, supporting the concept of charge balance in their TS and of concerted-associative pathways for biocatalysis. Above all, superposition of GS and TS structures reveals that in associative phosphoryl transfer, the phosphorus atom migrates through a triangle of three, near-stationary, equatorial oxygens. The extension of these studies to near attack conformers further illuminates enzyme catalysis of phosphoryl transfer.