Oxygen-insensitive nitroreductase E. coli NfsA, but not NfsB, is inhibited by fumarate.

Escherichia coli NfsA and NfsB are founding members of two flavoprotein families that catalyze the oxygen-insensitive reduction of nitroaromatics and quinones by NAD(P)H. This reduction is required for the activity of nitrofuran antibiotics and the enzymes have also been proposed for use with nitroaromatic prodrugs in cancer gene therapy and biocatalysis, but the roles of the proteins in vivo in bacteria are not known. NfsA is NADPH-specific whereas NfsB can also use NADH. The crystal structures of E. coli NfsA and NfsB and several analogs have been determined previously. In our crystal trials, we unexpectedly observed NfsA bound to fumarate. We here present the X-ray structure of the E. coli NfsA-fumarate complex and show that fumarate acts as a weak inhibitor of NfsA but not of NfsB. The structural basis of this differential inhibition is conserved in the two protein families and occurs at fumarate concentrations found in vivo, so impacting the efficacy of these proteins.

Structure and Dynamics of Three Escherichia coli NfsB Nitro-Reductase Mutants Selected for Enhanced Activity with the Cancer Prodrug CB1954.

Escherichia coli NfsB has been studied extensively for its potential for cancer gene therapy by reducing the prodrug CB1954 to a cytotoxic derivative. We have previously made several mutants with enhanced activity for the prodrug and characterised their activity in vitro and in vivo. Here, we determine the X-ray structure of our most active triple and double mutants to date, T41Q/N71S/F124T and T41L/N71S. The two mutant proteins have lower redox potentials than wild-type NfsB, and the mutations have lowered activity with NADH so that, in contrast to the wild-type enzyme, the reduction of the enzyme by NADH, rather than the reaction with CB1954, has a slower maximum rate. The structure of the triple mutant shows the interaction between Q41 and T124, explaining the synergy between these two mutations. Based on these structures, we selected mutants with even higher activity. The most active one contains T41Q/N71S/F124T/M127V, in which the additional M127V mutation enlarges a small channel to the active site. Molecular dynamics simulations show that the mutations or reduction of the FMN cofactors of the protein has little effect on its dynamics and that the largest backbone fluctuations occur at residues that flank the active site, contributing towards its broad substrate range.

The structures of E. coli NfsA bound to the antibiotic nitrofurantoin; to 1,4-benzoquinone and to FMN.

NfsA is a dimeric flavoprotein that catalyses the reduction in nitroaromatics and quinones by NADPH. This reduction is required for the activity of nitrofuran antibiotics. The crystal structure of free Escherichia coli NfsA and several homologues have been determined previously, but there is no structure of the enzyme with ligands. We present here crystal structures of oxidised E. coli NfsA in the presence of several ligands, including the antibiotic nitrofurantoin. Nitrofurantoin binds with the furan ring, rather than the nitro group that is reduced, near the N5 of the FMN. Molecular dynamics simulations show that this orientation is only favourable in the oxidised enzyme, while potentiometry suggests that little semiquinone is formed in the free protein. This suggests that the reduction occurs by direct hydride transfer from FMNH- to nitrofurantoin bound in the reverse orientation to that in the crystal structure. We present a model of nitrofurantoin bound to reduced NfsA in a viable hydride transfer orientation. The substrate 1,4-benzoquinone and the product hydroquinone are positioned close to the FMN N5 in the respective crystal structures with NfsA, suitable for reaction, but are mobile within the active site. The structure with a second FMN, bound as a ligand, shows that a mobile loop in the free protein forms a phosphate-binding pocket. NfsA is specific for NADPH and a similar conformational change, forming a phosphate-binding pocket, is likely to also occur with the natural cofactor.

Location-Dependent Lanthanide Selectivity Engineered into Structurally Characterized Designed Coiled Coils.

Herein we report unprecedented location-dependent, size-selective binding to designed lanthanide (Ln3+ ) sites within miniature protein coiled coil scaffolds. Not only do these engineered sites display unusual Ln3+ selectivity for moderately large Ln3+ ions (Nd to Tb), for the first time we demonstrate that selectivity can be location-dependent and can be programmed into the sequence. A 1 nm linear translation of the binding site towards the N-terminus can convert a selective site into a highly promiscuous one. An X-ray crystal structure, the first of a lanthanide binding site within a coiled coil to be reported, coupled with CD studies, reveal the existence of an optimal radius that likely stems from the structural constraints of the coiled coil scaffold. To the best of our knowledge this is the first report of location-dependent metal selectivity within a coiled coil scaffold, as well as the first report of location-dependent Ln3+ selectivity within a protein.

Identification of Phosphorylation Sites Altering Pollen Soluble Inorganic Pyrophosphatase Activity.

Protein phosphorylation regulates numerous cellular processes. Identifying the substrates and protein kinases involved is vital to understand how these important posttranslational modifications modulate biological function in eukaryotic cells. Pyrophosphatases catalyze the hydrolysis of inorganic phosphate (PPi) to inorganic phosphate Pi, driving biosynthetic reactions; they are essential for low cytosolic inorganic phosphate. It was suggested recently that posttranslational regulation of Family I soluble inorganic pyrophosphatases (sPPases) may affect their activity. We previously demonstrated that two pollen-expressed sPPases, Pr-p26.1a and Pr-p26.1b, from the flowering plant Papaver rhoeas were inhibited by phosphorylation. Despite the potential significance, there is a paucity of data on sPPase phosphorylation and regulation. Here, we used liquid chromatographic tandem mass spectrometry to map phosphorylation sites to the otherwise divergent amino-terminal extensions on these pollen sPPases. Despite the absence of reports in the literature on mapping phosphorylation sites on sPPases, a database survey of various proteomes identified a number of examples, suggesting that phosphorylation may be a more widely used mechanism to regulate these enzymes. Phosphomimetic mutants of Pr-p26.1a/b significantly and differentially reduced PPase activities by up to 2.5-fold at pH 6.8 and 52% in the presence of Ca2+ and hydrogen peroxide over unmodified proteins. This indicates that phosphoregulation of key sites can inhibit the catalytic responsiveness of these proteins in concert with key intracellular events. As sPPases are essential for many metabolic pathways in eukaryotic cells, our findings identify the phosphorylation of sPPases as a potential master regulatory mechanism that could be used to attenuate metabolism.

Phytoremediation Reduces Dust Emissions from Metal(loid)-Contaminated Mine Tailings.

Environmental and health risk concerns relating to airborne particles from mining operations have focused primarily on smelting activities. However, there are only three active copper smelters and less than a dozen smelters for other metals compared to an estimated 500000 abandoned and unreclaimed hard rock mine tailings in the US that have the potential to generate dust. The problem can also extend to modern tailings impoundments, which may take decades to build and remain barren for the duration before subsequent reclamation. We examined the impact of vegetation cover and irrigation on dust emissions and metal(loid) transport from mine tailings during a phytoremediation field trial at the Iron King Mine and Humboldt Smelter Superfund (IKMHSS) site. Measurements of horizontal dust flux following phytoremediation reveals that vegetated plots with 16% and 32% canopy cover enabled an average dust deposition of 371.7 and 606.1 g m-2 y-1, respectively, in comparison to the control treatment which emitted dust at an average rate of 2323 g m-2 y-1. Horizontal dust flux and dust emissions from the vegetated field plots are comparable to emission rates in undisturbed grasslands. Further, phytoremediation was effective at reducing the concentration of fine particulates, including PM1, PM2.5, and PM4, which represent the airborne particulates with the greatest health risks and the greatest potential for long-distance transport. This study demonstrates that phytoremediation can substantially decrease dust emissions as well as the transport of windblown contaminants from mine tailings.

Intrinsic disorder in the partitioning protein KorB persists after co-operative complex formation with operator DNA and KorA.

The ParB protein, KorB, from the RK2 plasmid is required for DNA partitioning and transcriptional repression. It acts co-operatively with other proteins, including the repressor KorA. Like many multifunctional proteins, KorB contains regions of intrinsically disordered structure, existing in a large ensemble of interconverting conformations. Using NMR spectroscopy, circular dichroism and small-angle neutron scattering, we studied KorB selectively within its binary complexes with KorA and DNA, and within the ternary KorA/KorB/DNA complex. The bound KorB protein remains disordered with a mobile C-terminal domain and no changes in the secondary structure, but increases in the radius of gyration on complex formation. Comparison of wild-type KorB with an N-terminal deletion mutant allows a model of the ensemble average distances between the domains when bound to DNA. We propose that the positive co-operativity between KorB, KorA and DNA results from conformational restriction of KorB on binding each partner, while maintaining disorder.

Flexibility of KorA, a plasmid-encoded, global transcription regulator, in the presence and the absence of its operator.

The IncP (Incompatibility group P) plasmids are important carriers in the spread of antibiotic resistance across Gram-negative bacteria. Gene expression in the IncP-1 plasmids is stringently controlled by a network of four global repressors, KorA, KorB, TrbA and KorC interacting cooperatively. Intriguingly, KorA and KorB can act as co-repressors at varying distances between their operators, even when they are moved to be on opposite sides of the DNA. KorA is a homodimer with the 101-amino acid subunits, folding into an N-terminal DNA-binding domain and a C-terminal dimerization domain. In this study, we have determined the structures of the free KorA repressor and two complexes each bound to a 20-bp palindromic DNA duplex containing its consensus operator sequence. Using a combination of X-ray crystallography, nuclear magnetic resonance spectroscopy, SAXS and molecular dynamics calculations, we show that the linker between the two domains is very flexible and the protein remains highly mobile in the presence of DNA. This flexibility allows the DNA-binding domains of the dimer to straddle the operator DNA on binding and is likely to be important in cooperative binding to KorB. Unexpectedly, the C-terminal domain of KorA is structurally similar to the dimerization domain of the tumour suppressor p53.

The specificity of proton-translocating transhydrogenase for nicotinamide nucleotides.

In its forward direction, transhydrogenase couples the reduction of NADP(+) by NADH to the outward translocation of protons across the membrane of bacteria and animal mitochondria. The enzyme has three components: dI and dIII protrude from the membrane and dII spans the membrane. Hydride transfer takes place between nucleotides bound to dI and dIII. Studies on the kinetics of a lag phase at the onset of a "cyclic reaction" catalysed by complexes of the dI and dIII components of transhydrogenase from Rhodospirillum rubrum, and on the kinetics of fluorescence changes associated with nucleotide binding, reveal two features. Firstly, the binding of NADP(+) and NADPH to dIII is extremely slow, and is probably limited by the conversion of the occluded to the open state of the complex. Secondly, dIII can also bind NAD(+) and NADH. Extrapolating to the intact enzyme this binding to the "wrong" site could lead to slip: proton translocation without change in the nucleotide redox state, which would have important consequences for bacterial and mitochondrial metabolism.

Response of key soil parameters during compost-assisted phytostabilization in extremely acidic tailings: effect of plant species.

Phytostabilization of mine tailings acts to mitigate both eolian dispersion and water erosion events which can disseminate barren tailings over large distances. This technology uses plants to establish a vegetative cover to permanently immobilize contaminants in the rooting zone, often requiring addition of an amendment to assist plant growth. Here we report the results of a greenhouse study that evaluated the ability of six native plant species to grow in extremely acidic (pH ∼ 2.5) metalliferous (As, Pb, Zn: 2000-3000 mg kg(-1)) mine tailings from Iron King Mine Humboldt Smelter Superfund site when amended with a range of compost concentrations. Results revealed that three of the six plant species tested (buffalo grass, mesquite, and catclaw acacia) are good candidates for phytostabilization at an optimum level of 15% compost (w/w) amendment showing good growth and minimal shoot accumulation of metal(loid)s. A fourth candidate, quailbush, also met all criteria except for exceeding the domestic animal toxicity limit for shoot accumulation of zinc. A key finding of this study was that the plant species that grew most successfully on these tailings significantly influenced key tailings parameters; direct correlations between plant biomass and both increased tailings pH and neutrophilic heterotrophic bacterial counts were observed. We also observed decreased iron oxidizer counts and decreased bioavailability of metal(loid)s mainly as a result of compost amendment. Taken together, these results suggest that the phytostabilization process reduced tailings toxicity as well as the potential for metal(loid) mobilization. This study provides practical information on plant and tailings characteristics that is critically needed for successful implementation of assisted phytostabilization on acidic, metalliferous mine tailings sites.

The 3D-structure, kinetics and dynamics of the E. coli nitroreductase NfsA with NADP+ provide glimpses of its catalytic mechanism.

Nitroreductases activate nitroaromatic antibiotics and cancer prodrugs to cytotoxic hydroxylamines and reduce quinones to quinols. Using steady-state and stopped-flow kinetics, we show that the Escherichia coli nitroreductase NfsA is 20-50 fold more active with NADPH than with NADH and that product release may be rate-limiting. The crystal structure of NfsA with NADP+ shows that a mobile loop forms a phosphate-binding pocket. The nicotinamide ring and nicotinamide ribose are mobile, as confirmed in molecular dynamics (MD) simulations. We present a model of NADPH bound to NfsA. Only one NADP+ is seen bound to the NfsA dimers, and MD simulations show that binding of a second NADP(H) cofactor is unfavourable, suggesting that NfsA and other members of this protein superfamily may have a half-of-sites mechanism.

Order and disorder in the domain organization of the plasmid partition protein KorB.

The plasmid partition protein KorB has a dual role: it is essential for the correct segregation of the low copy number broad host range RK2 plasmid while also being an important regulator of transcription. KorB belongs to the ParB family of proteins, and partitioning in RK2 has been studied as a simplified model of bacterial chromosome segregation. Structural information on full-length ParB proteins is limited, mainly due to the inability to grow crystals suitable for diffraction studies. We show, using CD and NMR, that KorB has regions of significant intrinsic disorder and hence it adopts a multiplicity of conformations in solution. The biophysical data are consistent with bioinformatic predictions based on the amino acid sequence that the N-terminal region and also the region between the central DNA-binding domain and the C-terminal dimerization domain are intrinsically disordered. We have used small angle x-ray scattering data to determine the ensemble of solution conformations for KorB and selected deletion mutants, based on models of the known domain structures. This conformational range of KorB is likely to be biologically required for DNA partitioning and for binding to a diverse set of partner proteins.

The homodimeric GBS1074 from Streptococcus agalactiae.

ESAT-6 is a well characterized secreted protein from Mycobacterium tuberculosis and represents the archetype of the WXG100 family of proteins. Genes encoding ESAT-6 homologues have been identified in the genome of the human pathogen Streptococcus agalactiae; one of these genes, esxA, has been cloned and the recombinant protein has been crystallized. In contrast to M. tuberculosis ESAT-6, the crystal structure of GBS1074 reveals a homodimeric structure similar to homologous structures from Staphylococcus aureus and Helicobacter pylori. Intriguingly, GBS1074 forms elongated fibre-like assemblies in the crystal structure.

Steady-state and stopped-flow kinetic studies of three Escherichia coli NfsB mutants with enhanced activity for the prodrug CB1954.

The enzyme nitroreductase, NfsB, from Escherichia coli has entered clinical trials for cancer gene therapy with the prodrug CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide]. However, CB1954 is a poor substrate for the enzyme. Previously we made several NfsB mutants that show better activity with CB1954 in a cell-killing assay in E. coli. Here we compare the kinetic parameters of wild-type NfsB with CB1954 to those of the most active single, double, and triple mutants isolated to date. For wild-type NfsB the global kinetic parameters for both k(cat) and K(m) for CB1954 are about 20-fold higher than previously estimated; however, the measured specificity constant, k(cat)/K(m) is the same. All of the mutants are more active with CB1954 than the wild-type enzyme, the most active mutant showing about 100-fold improved specificity constant with CB1954 over the wild-type protein with little effect on k(cat). This enhancement in specificity constants for the mutants is not seen with the antibiotic nitrofurazone as substrate, leading to reversed nitroaromatic substrate selectivity for the double and triple mutants. However, similar enhancements in specificity constants are found with the quinone menadione. Stopped-flow kinetic studies suggest that the rate-determining step of the reaction is likely to be the release of products. The most active mutant is also selective for the 4-nitro group of CB1954, rather than the 2-nitro group, giving the more cytotoxic reduction product. The double and triple mutants should be much more effective enzymes for use with CB1954 in prodrug-activation gene therapy.

Structure of rat odorant-binding protein OBP1 at 1.6 A resolution.

The nasal mucosa is a specialist interfacial region sandwiched between the olfactory system and the gaseous chemical milieu. In mammals and insects, this region is rich in odorant-binding proteins that are thought to aid olfaction by assisting mass transfer of the many different organoleptic compounds that make up the olfactory landscape. However, in mammals at least, our grasp on the exact function of odorant-binding proteins is tentative and better insight into the role of these proteins is warranted, not least because of their apparent significance in the olfactory systems of insects. Here, the crystal structure of rat odorant-binding protein 1 is reported at 1.6 A resolution. This protein is one of the best-characterized mammalian odorant-binding proteins and only the third such protein structure to be solved at high resolution. The protein was crystallized in the holo form and contains an unidentifiable ligand that is probably an artefact from the Pichia pastoris expression system. Comparisons are made between this structure and a modelled OBP1 structure produced using the crystal structure of aphrodisin as a template. Comparisons are also made between OBP1 and the other two rat OBP subtypes, for which crystallographic data are unavailable. Interestingly, we also show that OBP1 is monomeric, which is in contrast to its previous assignment.

Changes in lead and zinc lability during weathering-induced acidification of desert mine tailings: Coupling chemical and micro-scale analyses.

Desert mine tailings may accumulate toxic metals in the near surface centimeters because of low water through-flux rates. Along with other constraints, metal toxicity precludes natural plant colonization even over decadal time scales. Since unconsolidated particles can be subjected to transport by wind and water erosion, potentially resulting in direct human and ecosystem exposure, there is a need to know how the lability and form of metals change in the tailings weathering environment. A combination of chemical extractions, X-ray diffraction, micro-X-ray fluorescence spectroscopy, and micro-Raman spectroscopy were employed to study Pb and Zn contamination in surficial arid mine tailings from the Arizona Klondyke State Superfund Site. Initial site characterization indicated a wide range in pH (2.5 to 8.0) in the surficial tailings pile. Ligand-promoted (DTPA) extractions, used to assess plant-available metal pools, showed decreasing available Zn and Mn with progressive tailings acidification. Aluminum shows the inverse trend, and Pb and Fe show more complex pH dependence. Since the tailings derive from a common source and parent mineralogy, it is presumed that variations in pH and "bioavailable" metal concentrations result from associated variation in particle-scale geochemistry. Four sub-samples, ranging in pH from 2.6 to 5.4, were subjected to further characterization to elucidate micro-scale controls on metal mobility. With acidification, total Pb (ranging from 5 - 13 g kg(-1)) was increasingly associated with Fe and S in plumbojarosite aggregates. For Zn, both total (0.4 - 6 g kg(-1)) and labile fractions decreased with decreasing pH. Zinc was found to be primarily associated with the secondary Mn phases manjiroite and chalcophanite. The results suggest that progressive tailings acidification diminishes the overall lability of the total Pb and Zn pools.

Soil Microbiome Dynamics During Pyritic Mine Tailing Phytostabilization: Understanding Microbial Bioindicators of Soil Acidification.

Challenges to the reclamation of pyritic mine tailings arise from in situ acid generation that severely constrains the growth of natural revegetation. While acid mine drainage (AMD) microbial communities are well-studied under highly acidic conditions, fewer studies document the dynamics of microbial communities that generate acid from pyritic material under less acidic conditions that can allow establishment and support of plant growth. This research characterizes the taxonomic composition dynamics of microbial communities present during a 6-year compost-assisted phytostabilization field study in extremely acidic pyritic mine tailings. A complementary microcosm experiment was performed to identify successional community populations that enable the acidification process across a pH gradient. Taxonomic profiles of the microbial populations in both the field study and microcosms reveal shifts in microbial communities that play pivotal roles in facilitating acidification during the transition between moderately and highly acidic conditions. The potential co-occurrence of organoheterotrophic and lithoautotrophic energy metabolisms during acid generation suggests the importance of both groups in facilitating acidification. Taken together, this research suggests that key microbial populations associated with pH transitions could be used as bioindicators for either sustained future plant growth or for acid generation conditions that inhibit further plant growth.

The C-terminal tail of the bacterial translocation ATPase SecA modulates its activity.

In bacteria, the translocation of proteins across the cytoplasmic membrane by the Sec machinery requires the ATPase SecA. SecA binds ribosomes and recognises nascent substrate proteins, but the molecular mechanism of nascent substrate recognition is unknown. We investigated the role of the C-terminal tail (CTT) of SecA in nascent polypeptide recognition. The CTT consists of a flexible linker (FLD) and a small metal-binding domain (MBD). Phylogenetic analysis and ribosome binding experiments indicated that the MBD interacts with 70S ribosomes. Disruption of the MBD only or the entire CTT had opposing effects on ribosome binding, substrate-protein binding, ATPase activity and in vivo function, suggesting that the CTT influences the conformation of SecA. Site-specific crosslinking indicated that F399 in SecA contacts ribosomal protein uL29, and binding to nascent chains disrupts this interaction. Structural studies provided insight into the CTT-mediated conformational changes in SecA. Our results suggest a mechanism for nascent substrate protein recognition.

A New Optical Remote Sensing Technique for High-Resolution Mapping of Soil Moisture.

The recently developed OPtical TRApezoid Model (OPTRAM) has been successfully applied for watershed scale soil moisture (SM) estimation based on remotely sensed shortwave infrared (SWIR) transformed reflectance (TRSWIR) and the normalized difference vegetation index (NDVI). This study is aimed at the evaluation of OPTRAM for field scale precision agriculture applications using ultrahigh spatial resolution optical observations obtained with one of the world's largest field robotic phenotyping scanners located in Maricopa, Arizona. We replaced NDVI with the soil adjusted vegetation index (SAVI), which has been shown to be more accurate for cropped agricultural fields that transition from bare soil to dense vegetation cover. The OPTRAM was parameterized based on the trapezoidal geometry of the pixel distribution within the TRSWIR-SAVI space, from which wet- and dry-edge parameters were determined. The accuracy of the resultant SM estimates is evaluated based on a comparison with ground reference measurements obtained with Time Domain Reflectometry (TDR) sensors deployed to monitor surface, near-surface and root zone SM. The obtained results indicate an SM estimation error between 0.045 and 0.057 cm3 cm-3 for the near-surface and root zone, respectively. The high resolution SM maps clearly capture the spatial SM variability at the sensor locations. These findings and the presented framework can be applied in conjunction with Unmanned Aerial System (UAS) observations to assist with farm scale precision irrigation management to improve water use efficiency of cropping systems and conserve water in water-limited regions of the world.

The crystal structure of the cytosolic exopolyphosphatase from Saccharomyces cerevisiae reveals the basis for substrate specificity.

Inorganic long-chain polyphosphate is a ubiquitous linear polymer in biology, consisting of many phosphate moieties linked by phosphoanhydride bonds. It is synthesized by polyphosphate kinase, and metabolised by a number of enzymes, including exo- and endopolyphosphatases. The Saccharomyces cerevisiae gene PPX1 encodes for a 45 kDa, metal-dependent, cytosolic exopolyphosphatase that processively cleaves the terminal phosphate group from the polyphosphate chain, until inorganic pyrophosphate is all that remains. PPX1 belongs to the DHH family of phosphoesterases, which includes: family-2 inorganic pyrophosphatases, found in Gram-positive bacteria; prune, a cyclic AMPase; and RecJ, a single-stranded DNA exonuclease. We describe the high-resolution X-ray structures of yeast PPX1, solved using the multiple isomorphous replacement with anomalous scattering (MIRAS) technique, and its complexes with phosphate (1.6 A), sulphate (1.8 A) and ATP (1.9 A). Yeast PPX1 folds into two domains, and the structures reveal a strong similarity to the family-2 inorganic pyrophosphatases, particularly in the active-site region. A large, extended channel formed at the interface of the N and C-terminal domains is lined with positively charged amino acids and represents a conduit for polyphosphate and the site of phosphate hydrolysis. Structural comparisons with the inorganic pyrophosphatases and analysis of the ligand-bound complexes lead us to propose a hydrolysis mechanism. Finally, we discuss a structural basis for substrate selectivity and processivity.