Gene Coexpression Connectivity Predicts Gene Targets Underlying High Ionic-Liquid Tolerance in Yarrowia lipolytica.

Microbial tolerance to organic solvents such as ionic liquids (ILs) is a robust phenotype beneficial for novel biotransformation. While most microbes become inhibited in 1% to 5% (vol/vol) IL (e.g.

CASPER: An Integrated Software Platform for Rapid Development of CRISPR Tools.

Both academic and enterprise software solutions exist for designing CRISPR targets. They offer advantages when designing guide RNAs (gRNAs) but often focus on a select number of model organisms. Those that offer a wide variety of organisms can be limited in support of alternative endonucleases and downstream analyses such as multitargeting and population analyses to interrogate a microbiome.

Kβ X-ray Emission Spectroscopy as a Probe of Cu(I) Sites: Application to the Cu(I) Site in Preprocessed Galactose Oxidase.

Cu(I) active sites in metalloproteins are involved in O activation, but their O reactivity is difficult to study due to the Cu(I) d closed shell which precludes the use of conventional spectroscopic methods. Kβ X-ray emission spectroscopy (XES) is a promising technique for investigating Cu(I) sites as it detects photons emitted by electronic transitions from occupied orbitals. Here, we demonstrate the utility of Kβ XES in probing Cu(I) sites in model complexes and a metalloprotein.

Structure of the Reduced Copper Active Site in Preprocessed Galactose Oxidase: Ligand Tuning for One-Electron O Activation in Cofactor Biogenesis.

Galactose oxidase (GO) is a copper-dependent enzyme that accomplishes 2e substrate oxidation by pairing a single copper with an unusual cysteinylated tyrosine (Cys-Tyr) redox cofactor. Previous studies have demonstrated that the post-translational biogenesis of Cys-Tyr is copper- and O-dependent, resulting in a self-processing enzyme system. To investigate the mechanism of cofactor biogenesis in GO, the active-site structure of Cu(I)-loaded GO was determined using X-ray absorption near edge structure (XANES) and extended X-ray absorption fine structure (EXAFS) spectroscopy, and density-functional theory (DFT) calculations were performed on this model.

Inhibition and oxygen activation in copper amine oxidases.

Copper-containing amine oxidases (CuAOs) use both copper and 2,4,5-trihydroxyphenylalanine quinone (TPQ) to catalyze the oxidative deamination of primary amines. The CuAO active site is highly conserved and comprised of TPQ and a mononuclear type II copper center that exhibits five-coordinate, distorted square pyramidal coordination geometry with histidine ligands and equatorially and axially bound water in the oxidized, resting state. The active site is buried within the protein, and CuAOs from various sources display remarkable diversity with respect to the composition of the active site channel and cofactor accessibility.

Combined deficiencies of vitamins b1 and C in well-nourished patients.

Vitamin deficiencies such as scurvy and Wernicke's encephalopathy are poorly recognized in Western populations. Inadequate intake of these micronutrients and low levels when measured are more frequent than expected. Cases of combined vitamin deficiencies such as that of ascorbic acid and thiamine are seldom reported.

Experimental and computational evidence of metal-O2 activation and rate-limiting proton-coupled electron transfer in a copper amine oxidase.

The mechanism of O(2) reduction by copper amine oxidase from Arthrobacter globiformus (AGAO) is analyzed in relation to the cobalt-substituted protein. The enzyme utilizes a tyrosine-derived topaquinone cofactor to oxidize primary amines and reduce O(2) to H(2)O(2). Steady-state kinetics indicate that amine-reduced CuAGAO is reoxidized by O(2) >10(3) times faster than the CoAGAO analogue.

Direct electron transfer from pseudoazurin to nitrous oxide reductase in catalytic N2O reduction.

Pseudoazurin (PAz), a well-characterized blue copper electron-transfer protein, is shown herein to be capable of mediating electron transfer to the nitrous oxide reductase (N(2)OR) from Achromobacter cycloclastes (Ac). Spectroscopic measurements demonstrate that reduced PAz is efficiently re-oxidized by a catalytic amount of N(2)OR in the presence of N(2)O. Fits of the kinetics resulted in K(M) (N(2)O) and k(cat) values of 19.

Role of the Tyr-Cys cross-link to the active site properties of galactose oxidase.

The catalytically relevant, oxidized state of the active site [Cu(II)-Y·-C] of galactose oxidase (GO) is composed of antiferromagnetically coupled Cu(II) and a post-translationally generated Tyr-Cys radical cofactor [Y·-C]. The thioether bond of the Tyr-Cys cross-link has been shown experimentally to affect the stability, the reduction potential, and the catalytic efficiency of the GO active site. However, the origin of these structural and energetic effects on the GO active site has not yet been investigated in detail.

Cobalt substitution supports an inner-sphere electron transfer mechanism for oxygen reduction in pea seedling amine oxidase.

Copper amine oxidases (CAOs) are a large family of proteins that use molecular oxygen to oxidize amines to aldehydes with the concomitant production of hydrogen peroxide and ammonia. CAOs utilize two cofactors for this reaction: topaquinone (TPQ) and a Cu(II) ion. Two mechanisms for oxygen reduction have been proposed for these enzymes.

Amperometric measurement of glutamate release modulation by gabapentin and pregabalin in rat neocortical slices: role of voltage-sensitive Ca2+ α2δ-1 subunit.

Gabapentin (GBP; Neurontin) and pregabalin (PGB; Lyrica, S-(+)-3-isobutylgaba) are used clinically to treat several disorders associated with excessive or inappropriate excitability, including epilepsy; pain from diabetic neuropathy, postherpetic neuralgia, and fibromyalgia; and generalized anxiety disorder. The molecular basis for these drugs' therapeutic effects are believed to involve the interaction with the auxiliary α(2)δ subunit of voltage-sensitive Ca(2+) channel (VSCC) translating into a modulation of pathological neurotransmitter release. Glutamate as the primary excitatory neurotransmitter in the mammalian central nervous system contributes, under conditions of excessive glutamate release, to neurological and psychiatric disorders.

Spectroscopic identification of heme axial ligands in HtsA that are involved in heme acquisition by Streptococcus pyogenes.

The heme-binding proteins Shp and HtsA of Streptococcus pyogenes are part of the heme acquisition machinery in which Shp directly transfers its heme to HtsA. Mutagenesis and spectroscopic analyses were performed to identify the heme axial ligands in HtsA and to characterize axial mutants of HtsA. Replacements of the M79 and H229 residues, not the other methionine and histidine residues, with alanine convert UV-vis spectra of HtsA with a low-spin, hexacoordinate heme iron into spectra of high-spin heme complexes.

A new crystal form of human diamine oxidase.

Copper amine oxidases (CAOs) are ubiquitous in nature and catalyse the oxidative deamination of primary amines to the corresponding aldehydes. Humans have three viable CAO genes (AOC1-3). AOC1 encodes human diamine oxidase (hDAO), which is the frontline enzyme for histamine metabolism.

Non-covalent interactions in blue copper protein probed by Met16 mutation and electronic and resonance Raman spectroscopy of Achromobacter cycloclastes pseudoazurin.

We have used low-temperature (77K) resonance Raman (RR) spectroscopy as a probe of the electronic and molecular structure to investigate weak pi-pi interactions between the metal ion-coordinated His imidazoles and aromatic side chains in the second coordination sphere of blue copper proteins. For this purpose, the RR spectra of Met16 mutants of Achromobacter cycloclastes pseudoazurin (AcPAz) with aromatic (Met16Tyr, Met16Trp, and Met16Phe) and aliphatic (Met16Ala, Met16Val, Met16Leu, and Met16Ile) amino acid side chains have been obtained and analyzed over the 100-500cm(-1) spectral region. Subtle strengthening of the Cu(II)-S(Cys) interaction on replacing Met16 with Tyr, Trp, and Phe is indicated by the upshifted (0.

Structure and inhibition of human diamine oxidase.

Humans have three functioning genes that encode copper-containing amine oxidases. The product of the AOC1 gene is a so-called diamine oxidase (hDAO), named for its substrate preference for diamines, particularly histamine. hDAO has been cloned and expressed in insect cells and the structure of the native enzyme determined by X-ray crystallography to a resolution of 1.

Income volatility and psychological depression.

Income volatility appears to be increasing especially among lower income workers. Such volatility may reflect the ongoing shift of economic risk from employers to employees as marked by decreasing job security and employer-provided benefits. This study tests whether absolute volatility or downward volatility in income predict depression controlling for prior depression.

Kinetics and spectroscopic evidence that the Cu(I)-semiquinone intermediate reduces molecular oxygen in the oxidative half-reaction of Arthrobacter globiformis amine oxidase.

The role of copper during the reoxidation of substrate-reduced amine oxidases by O(2) has not yet been definitively established. Both outer-sphere and inner-sphere pathways for the reduction of O(2) to H(2)O(2) have been proposed. A key step in the inner-sphere mechanism is the reaction of O(2) directly with the Cu(I) center of a Cu(I)-semiquinone intermediate.

Cross-link formation of the cysteine 228-tyrosine 272 catalytic cofactor of galactose oxidase does not require dioxygen.

Galactose oxidase (GO) belongs to a class of proteins that self-catalyze assembly of their redox-active cofactors from active site amino acids. Generation of enzymatically active GO appears to require at least four sequential post-translational modifications: cleavage of a secretion signal sequence, copper-dependent cleavage of an N-terminal pro sequence, copper-dependent formation of a C228-Y272 thioether bond, and generation of the Y272 radical. The last two processes were investigated using a truncated protein (termed premat-GO) lacking the pro sequence and purified under copper-free conditions.

Complexes of the copper-containing amine oxidase from Arthrobacter globiformis with the inhibitors benzylhydrazine and tranylcypromine.

Complexes of Arthrobacter globiformis amine oxidase (AGAO) with the inhibitors benzylhydrazine and tranylcypromine (an antidepressant drug) have been refined at 1.86 and 1.65 A resolution, respectively.

Inner-sphere mechanism for molecular oxygen reduction catalyzed by copper amine oxidases.

Copper and topaquinone (TPQ) containing amine oxidases utilize O2 for the metabolism of biogenic amines while concomitantly generating H2O2 for use by the cell. The mechanism of O2 reduction has been the subject of long-standing debate due to the obscuring influence of a proton-coupled electron transfer between the tyrosine-derived TPQ and copper, a rapidly established equilibrium precluding assignment of the enzyme in its reactive form. Here, we show that substrate-reduced pea seedling amine oxidase (PSAO) exists predominantly in the Cu(I), TPQ semiquinone state.

The NIOSH Radiation Dose Reconstruction Project: managing technical challenges.

Approximately two years after promulgation of the Energy Employees Occupational Illness Compensation Program Act, the National Institute for Occupational Safety and Health Office of Compensation and Analysis Support selected a contractor team to perform many aspects of the radiation dose reconstruction process. The project scope and schedule necessitated the development of an organization involving a comparatively large number of health physicists. From the initial stages, there were many technical and managerial challenges that required continuous planning, integration, and conflict resolution.