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

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.

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 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.

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

Herein we report unprecedented location-dependent, size-selective binding to designed lanthanide (Ln ) sites within miniature protein coiled coil scaffolds. Not only do these engineered sites display unusual Ln selectivity for moderately large Ln 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.

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.

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 (TR) 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.

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

Challenges to the reclamation of pyritic mine tailings arise from 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.

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.

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.

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.

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 (PP) to inorganic phosphate P, driving biosynthetic reactions; they are essential for low cytosolic inorganic phosphate.

Phytostabilization of mine tailings using compost-assisted direct planting: Translating greenhouse results to the field.

Standard practice in reclamation of mine tailings is the emplacement of a 15 to 90cm soil/gravel/rock cap which is then hydro-seeded. In this study we investigate compost-assisted direct planting phytostabilization technology as an alternative to standard cap and plant practices. In phytostabilization the goal is to establish a vegetative cap using native plants that stabilize metals in the root zone with little to no shoot accumulation.

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.

Mechanism of intermediate filament recognition by plakin repeat domains revealed by envoplakin targeting of vimentin.

Plakin proteins form critical connections between cell junctions and the cytoskeleton; their disruption within epithelial and cardiac muscle cells cause skin-blistering diseases and cardiomyopathies. Envoplakin has a single plakin repeat domain (PRD) which recognizes intermediate filaments through an unresolved mechanism. Herein we report the crystal structure of envoplakin's complete PRD fold, revealing binding determinants within its electropositive binding groove.

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.

Isoleucine 259 and isoleucine 260 residues in Streptococcus gordonii soluble inorganic pyrophosphatase play an important role in enzyme activity.

The hinge region of the family II soluble inorganic pyrophosphatase (PPase) found in Streptococcus gordonii DL1 (Challis) has previously been shown to play an important role in opening and closing of the active site between the N- and C-terminal domains of PPase. Amino acid residues isoleucine 259 (I259) and isoleucine 260 (I260) are highly conserved among catalytically active family II PPases and are located very close to the hinge region. Substitution of either I259 or I260 with a hydrophilic acidic amino acid (glutamate or aspartate) resulted in adverse effects on the kinetic properties of the enzyme.

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.

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.

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.

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.

Characterization of two novel aldo-keto reductases from Arabidopsis: expression patterns, broad substrate specificity, and an open active-site structure suggest a role in toxicant metabolism following stress.

Aldo-keto reductases (AKRs) are widely distributed in nature and play numerous roles in the metabolism of steroids, sugars, and other carbonyls. They have also frequently been implicated in the metabolism of exogenous and endogenous toxicants, including those stimulated by stress. Although the Arabidopsis genome includes at least 21 genes with the AKR signature, very little is known of their functions.

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.

Mutations of key hydrophobic surface residues of 11 beta-hydroxysteroid dehydrogenase type 1 increase solubility and monodispersity in a bacterial expression system.

11 beta-Hydroxysteroid dehydrogenase type 1 (11 beta-HSD1) is a key enzyme in the conversion of cortisone to the functional glucocorticoid hormone cortisol. This activation has been implicated in several human disorders, notably the metabolic syndrome where 11 beta-HSD1 has been identified as a novel target for potential therapeutic drugs. Recent crystal structures have revealed the presence of a pronounced hydrophobic surface patch lying on two helices at the C-terminus.

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.

Differential specific radiation damage in the Cu II-bound and Pd II-bound forms of an alpha-helical foldamer: a case study of crystallographic phasing by RIP and SAD.

The high photon flux at third-generation synchrotron sources can inflict significant primary radiation damage upon macromolecular crystals, even when the crystals are cryocooled. However, specific radiation-induced structural changes can be exploited for de novo phasing by an approach known as radiation damage-induced phasing (RIP). Here, RIP and single-wavelength anomalous dispersion (SAD) phasing were alternatively used to derive experimental phases to 1.