Momentum-resolved evolution of the Kondo lattice into "hidden order" in URu2Si2.

We study, using high-resolution angle-resolved photoemission spectroscopy, the evolution of the electronic structure in URu2Si2 at the Γ, Z, and X high-symmetry points from the high-temperature Kondo-screened regime to the low-temperature hidden-order (HO) state. At all temperatures and symmetry points, we find structures resulting from the interaction between heavy and light bands related to the Kondo-lattice formation. At the X point, we directly measure a hybridization gap of 11 meV already open at temperatures above the ordered phase. Strikingly, we find that while the HO induces pronounced changes at Γ and Z, the hybridization gap at X does not change, indicating that the hidden-order parameter is anisotropic. Furthermore, at the Γ and Z points, we observe the opening of a gap in momentum in the HO state, and show that the associated electronic structure results from the hybridization of a light electron band with the Kondo-lattice bands characterizing the paramagnetic state.

Coherent heavy quasiparticles in a CePt5 surface alloy.

We report on the results of a high-resolution angle-resolved photoemission study on the ordered surface alloy CePt(5). The temperature dependence of the spectra show the formation of the coherent low-energy heavy-fermion band near the Fermi level. These experimental data are supported by a multiband model calculation in the framework of the dynamical mean-field theory.

Heterologous expression and characterization of choline oxidase from the soil bacterium Arthrobacter nicotianae.

In the course of a microbial screening of soil samples for new oxidases, different enrichment strategies were carried out. With choline as the only carbon source, a microorganism was isolated and identified as Arthrobacter nicotianae. From this strain, a gene coding for a choline oxidase was isolated from chromosomal DNA. This gene named codA was cloned in Escherichia coli BL21-Gold and the protein (An_CodA) heterologously overexpressed as a soluble intracellular protein of 59.1 kDa. Basic biochemical characterization of purified protein revealed a pH optimum of 7.4 and activity over a broad temperature range (15-70 degrees C). Specific activities were determined toward choline chloride (4.70 +/- 0.12 U/mg) and the synthetic analogs bis(2-hydroxyethyl)-dimethylammonium chloride (0.05 +/- 0.45 x 10(-2) U/mg) and tris-(2-hydroxyethyl)-methylammonium methylsulfate (0.01 +/- 0.12 x 10(-2) U/mg). With increasing number of oxidizable groups, a significant decrease in activity was noted. Determination of kinetic parameters in atmorspheric oxygen resulted in K (M) = 1.51 +/- 0.09 mM and V (max) = 42.73 +/- 0.42 mU/min for choline chloride and K (M) = 4.77 +/- 0.76 mM and V (max) = 48.40 +/- 2.88 mU/min for the reaction intermediate betaine aldehyde respectively. Nuclear magnetic resonance spectroscopic analysis of the products formed during the enzyme reaction with choline chloride showed that in vitro the intermediate betaine aldehyde exists also free in solution.

A novel screening assay for hydroxynitrile lyases suitable for high-throughput screening.

Hydroxynitrile lyases (Hnls) are important biocatalysts for the synthesis of optically pure cyanohydrins, which are used as precursors and building blocks for a wide range of high price fine chemicals. Although two Hnl enzymes, from the tropical rubber tree Hevea brasiliensis and from the almond tree Prunus amygdalus, are already used for large scale industrial applications, the enzymes still need to be improved and adapted to the special demands of industrial processes. In many cases directed evolution has been the method of choice to improve enzymes, which are applied as industrial biocatalysts. The screening procedure is the most crucial point in every directed evolution experiment. Herein, we describe the successful development of a novel screening assay for Hnls and its application in high-throughput screening of Escherichia coli mutant libraries. The new assay allows rapid screening of mutant libraries and facilitates the discovery of improved enzyme variants. Hnls catalyze the cleavage of cyanohydrins to hydrocyanic acid and the corresponding aldehyde or ketone. The enzyme assay is based on the detection of hydrocyanic acid produced, making it an all-purpose screening assay, without restriction to any kind of substrate. The gaseous HCN liberated within the Hnl reaction is detected by a visible colorimetric reaction. The facile, highly sensitive and reproducible screening method was validated by identifying new enzyme variants with novel substrate specificities.

Mechanism of cyanogenesis: the crystal structure of hydroxynitrile lyase from Hevea brasiliensis.

BACKGROUND: Over three thousand species of plants, including important food crops such as cassava, use cyanogenesis, the liberation of HCN upon tissue damage, as a defense against predation. Detoxification of cyanogenic food crops requires disruption of the cyanogenic pathway. Hydroxynitrile lyase is one of the key enzymes in cyanogenesis, catalyzing the decomposition of an alpha-cyanohydrin to form HCN plus the corresponding aldehyde or ketone. These enzymes are also of potential utility for industrial syntheses of optically pure chiral cyanohydrins, being used to catalyze the reverse reaction. We set out to gain insight into the catalytic mechanism of this important class of enzymes by determining the three-dimensional structure of hydroxynitrile lyase from the rubber tree, Hevea brasiliensis. RESULTS: The crystal structure of the enzyme has been determined to 1.9 A resolution. It belongs to the alpha/beta hydrolase superfamily, with an active site that is deeply buried within the protein and connected to the outside by a narrow tunnel. The catalytic triad is made up of Ser80, His235 and Asp207. By analogy with known mechanisms of other members of this superfamily, catalysis should involve an oxyanion hole formed by the main chain NH of Cys81 and the side chains of Cys81 and Thr11. Density attributed to a histidine molecule or ion is found in the active site. CONCLUSIONS: By analogy with other alpha/beta hydrolases, the reaction catalyzed by hydroxynitrile lyase involves a tetrahedral hemiketal or hemiacetal intermediate formed by nucleophilic attack of Ser80 on the substrate, stabilized by the oxyanion hole. The SH group of Cys81 is probably involved in proton transfer between the HCN and the hydroxynitrile OH. This mechanism is significantly different from the corresponding uncatalyzed solution reaction.

Interactions of cardiac glycosides with cultured cardiac cells. II. Biochemical and electron microscopic studies on the effects of ouabain on muscle and non-muscle cells.

Electron microscopic and biochemical studies revealed a salient difference in the response to toxic doses of ouabain by cultured cardiac muscle and non-muscle cells from neonatal rats. Progressive cellular injury in myocytes incubated with 1 . 10(-4)--1 . 10(-3) M ouabain ultimately leads to swelling and necrosis. The morphological damage in myocytes was accompanied by a drastic decrease in 14CO2 formation from 14C-labeled stearate or acetate but not glucose. Neither morphological nor biochemical impairments were observed in non-muscle cells. The interaction between ouabain and the cultured cells, using therapeutic doses of ouabain (i.e., less than 1 . 10(-7) M), was characterized. Two binding sites were described in both classes of cells, one site is a saturable K+-sensitive site whereas the other is non-saturable and K+-insensitive. The complexes formed between the sarcolemma receptor(s) and ouabain, at low concentrations of the drug (e.g., 7.52 . 10(-9) M), had Kd values of 8.9 . 10(-8) and 2.3 . 10(-8) M for muscle and non-muscle cells, respectively. The formation and dissociation of the complexes were affected by temperature and potassium ions.

Complete nucleotide sequence of Birmingham IncP alpha plasmids. Compilation and comparative analysis.

The IncP alpha promiscuous plasmid (R18, R68, RK2, RP1 and RP4) comprises 60,099 bp of nucleotide sequence, encoding at least 74 genes. About 40 kb of the genome, designated the IncP core and including all essential replication and transfer functions, can be aligned with equivalent sequences in the IncP beta plasmid R751. The compiled IncP alpha sequence revealed several previously unidentified reading frames that are potential genes. IncP alpha plasmids carry genetic information very efficiently: the coding sequences of the genes are closely packed but rarely overlap, and occupy almost 86% of the genome's nucleotide sequence. All of the 74 genes should be expressed, although there is as yet experimental evidence for expression of only 60 of them. Six examples of tandem-in-frame initiation sites specifying two gene products each are known. Two overlapping gene arrangements occupy different reading frames of the same region. Intergenic regions include most of the 25 promoters; transcripts are usually polycistronic. Translation of most of the open reading frames seems to be initiated independently, each from its own ribosomal binding and initiation site, although, a few cases of coupled translation have been reported. The most frequently used initiation codon is AUG but translation for a few open reading frames begins at GUG or UUG. The most common stop-codon is UGA followed by UAA and then UAG. Regulatory circuits are complex and largely dependent on two components of the central control operon. KorA and KorB are transcriptional repressors controlling at least seven operons. KorA and KorB act synergistically in several cases by recognizing and binding to conserved nucleotide sequences. Twelve KorB binding sites were found around the IncP alpha sequence and these are conserved in R751 (IncP beta) with respect to both sequence and location. Replication of IncP alpha plasmids requires oriV and the plasmid-encoded initiator protein TrfA in combination with the host-encoded replication machinery. Conjugative plasmid transfer depends on two separate regions occupying about half of the genome. The primary segregational stability system designated Par/Mrs consists of a putative site-specific recombinase, a possible partitioning apparatus and a post-segregational lethality mechanism, all encoded in two divergent operons. Proteins related to the products of F sop and P1 par partitioning genes are separately encoded in the central control operon.

The synthesis of chiral cyanohydrins by oxynitrilases.

Enantiomerically pure cyanohydrins are important synthetic intermediates for pharmaceuticals and agrochemicals. They are produced by enzyme-catalysed synthesis using oxynitrilases. Sufficient quantities of enzyme are available via cheap natural sources and there have been recent advances in overexpression production of cyanohydrins on an industrial scale.

Structure of the xylanase from Penicillium simplicissimum.

Despite its relatively low pH and temperature optimum, the xylanase from Penicillium simplicissimum performs exceedingly well under conditions of paper bleaching. We have purified and characterized this enzyme, which belongs to family 10 of glycosyl hydrolases. Its gene was cloned, and the sequence of the protein was deduced from the nucleotide sequence. The xylanase was crystallized from ammonium sulfate at pH 8.4, and X-ray data were collected at cryo-temperature to a crystallographic resolution of 1.75 A. The crystal structure was solved by molecular replacement using the catalytic domain of the Clostridium thermocellum xylanase as a search model, and refined to a residual of R = 20% (R(free) = 23%) for data between 10 and 1.75 A. The xylanase folds in an (alpha/beta)8 barrel (TIM-barrel), with additional helices and loops arranged at the "top" forming the active site cleft. In its overall shape, the P. simplicissimum xylanase structure is similar to other family 10 xylanases, but its active site cleft is much shallower and wider. This probably accounts for the differences in catalysis and in the mode of action of this enzyme. Three glycerol molecules were observed to bind within the active site groove, one of which interacts directly with the catalytic glutamate residues. It appears that they occupy putative xylose binding subsites.

Comparison of hypothalamus-pituitary-adrenal axis suppression from superpotent topical steroids by standard endocrine function testing and gas chromatographic mass spectrometry.

We evaluated 38 males who had psoriasis vulgaris for evidence of hypothalamus-pituitary-adrenal axis suppression (HPAS) during treatment with superpotent topical glucocorticosteroids. All men were treated with 49 g per week of either Betamethasone Diproprionate in an optimized vehicle or Clobetasol Proprionate ointment. Three methods used to assess HPAS were compared. Classic 8 a.m. plasma cortisol measurements, urinary-free cortisol, and 17-hydroxycorticosteroid determinations and gas chromatograph-mass spectrometry (GCMS) quantitation of urinary cortisol metabolites were compared. Values for all methods were obtained just prior to therapy and at days 4, 7, 14, and 21 during therapy and at day 28 after treatment was stopped for 7 d. Plasma cortisol measurements correlated well with other measures of HPAS. GCMS determination of urinary cortisol metabolites was slightly more sensitive at detecting HPAS than the other two methods. Persistent HPAS after day 7 was only appreciated by GCMS. Urinary-free cortisol and 17-hydroxycortisol was the least sensitive of the three methods. Analysis of urinary cortisol metabolites by GCMS may be most useful in the monitoring of HPAS resulting from use of topical glucocorticosteroid preparations.

Efficient secretion of Bacillus subtilis levanase by Saccharomyces cerevisiae.

The secretion of Bacillus subtilis (Bs) levanase (Lev) was studied in the yeast Saccharomyces cerevisiae. A set of different yeast expression plasmids, based on the constitutive PGK promoter and harbouring the Bs Lev-encoding gene (sacC), was constructed. In these plasmids, the original Bs signal sequence was either intact, partially deleted or entirely missing. With all constructs, Lev was produced from yeast transformants. However, only when the intact bacterial signal peptide was present was the synthesized enzyme secreted; around 20% was found in the periplasm and 30% in the culture medium. The secreted protein found in the periplasmic space was mainly core-glycosylated and unglycosylated, and had a size of 80-90 and 74 kDa, respectively. In contrast, Lev found in the culture medium was mainly hyper-glycosylated and had a size of 180-200 kDa. Yeast transformants harbouring sacC, but lacking parts of the bacterial signal sequence, only produced cytoplasmic protein which was not glycosylated and had a size of about 74 kDa. The deletion of the entire signal peptide and a further 22 amino acids at the N terminus of mature Lev resulted in a 71-kDa cytoplasmic protein which was not active.

Transformation of Penicillium chrysogenum using dominant selection markers and expression of an Escherichia coli lacZ fusion gene.

An industrial Penicillium chrysogenum strain was transformed using two dominant selection markers, namely the bacterial gene for phleomycin resistance (ble) fused to a fungal promoter, and the acetamidase (amdS) gene from Aspergillus nidulans. Transformation frequencies of up to 20 transformants per microgram of DNA were obtained with the ble system. With the amdS marker the frequency was up to 120 transformants. Cotransformation was very efficient when using amdS as a selection marker. The introduction of pAN5-41B, a plasmid carrying the Escherichia coli lacZ gene fused to the strong glyceraldehyde-3-phosphate dehydrogenase gene (gpd) promoter from A. nidulans, resulted in the formation of blue colonies on XGal plates indicating expression of the lacZ fusion gene in P. chrysogenum. A more detailed analysis of expression levels in several transformants showed that up to 6% of the total amount of soluble protein consists of the beta-galactosidase fusion protein.

Expression of the Bacillus subtilis levanase gene in Escherichia coli and Saccharomyces cerevisiae.

The gene coding for the inulin hydrolyzing enzyme levanase which was previously cloned from Bacillus subtilis was fused to the tac-promoter. Overexpression in Escherichia coli resulted in high amounts of intracellularly produced levanase (up to 20 U mg-1). After removal of the bacterial 5' sequences, the levanase gene was also cloned into a yeast expression vector based on the PGK-promoter. Clones containing the intact levanase gene including the bacterial signal sequence gave rise to synthesis of active levanase by Saccharomyces cerevisiae transformants. A considerable amount of levanase protein was found in the culture medium (around 0.5 U ml-1) indicating efficient secretion of B. subtilis levanase from yeast.

Molecular cloning, sequencing and expression in Escherichia coli of the poly(3-hydroxyalkanoate) synthesis genes from Alcaligenes latus DSM1124.

Fragments of chromosomal DNA from Alcaligenes latus DSM1124 were cloned into Escherichia coli and transformants were screened for poly(D(-)-3-hydroxybutyrate) [P(3HB)] production during excess carbon supply. A plasmid harboring a 5.5-kb insert of A. latus DNA was isolated from a P(3HB)-producing bacterial colony. The insert was partially sequenced and three major open reading frames (ORFs) were found, representing the PHA synthase (phaC), beta-ketothiolase (phaA) and acetoacetyl-CoA reductase (phaB) genes. They show striking homology to the Ralstonia eutropha (formerly Alcaligenes eutrophus) phaC (71%), phaA (77%) and phaB (80%) genes, and are organized in the same way. The only major difference is the replacement of 560 nucleotides by 160 non-homologous nucleotides in the 5' region of phaC in A. latus. The phaC ORF lacks 29 amino acids at the N-terminus, compared to that of R. eutropha, and starts with a GTG codon. The transcription start points of the operon were determined. P(3HB) production of recombinant E. coli strains harboring the pha operons of A. latus DSM1124 or R. eutropha H16 was investigated. Both operons gave rise to less than 5% P(3HB) formation during exponential growth. At the end of the growth phase, the P(3HB) content reached approximately 20% of cell dry mass. Under nitrogen-depleted conditions, the A. latus pha genes gave rise to 50-52% P(3HB), compared to 33-38% for the R. eutropha pha genes. No NADH oxidase activity was detectable in A. latus, indicating an impaired respiratory pathway and a dependence on PHA synthesis for storing reduction equivalents during growth.

A novel esterase from Burkholderia gladioli which shows high deacetylation activity on cephalosporins is related to beta-lactamases and DD-peptidases.

The gene (estB) encoding for a novel esterase (EstB) from Burkholderia gladioli (formerly Pseudomonas marginata) NCPPB 1891 was cloned in Escherichia coli. Sequence analysis showed an open reading frame encoding a polypeptide of 392 amino acid residues, with a molecular mass of about 42 kDa. Comparison of the amino acid sequence with those of other homologous enzymes indicated homologies to beta-lactamases, penicillin binding proteins and DD-peptidases. The serine residue (Ser(75)) which is located within a present class A beta-lactamase motif ([F,Y]-X-[L,I,V,M,F,Y]-X-S-[T,V]-X-K-X-X-X-X-[A,G,L]-X-X-[L,C]) was identified by site-directed mutagenesis to represent the active nucleophile. A second serine residue (Ser(149)) which is located within a G-x-S-x-G motif which is typically found in esterases and lipases was demonstrated not to play a significant role in enzyme function. The estB gene was overexpressed in E. coli using a tac promoter-based expression system. Investigation of EstB protein with respect to the ability to hydrolyse beta-lactam substrates clearly demonstrated that this protein has no beta-lactamase activity. The recombinant enzyme is active on triglycerides and on nitrophenyl esters with acyl chain lengths up to C6. The preference for short chain length substrates indicated that EstB is a typical carboxylesterase. As a special feature EstB esterase was found to have high deacetylation activity on cephalosporin derivatives.

Stability of r-microbes: stabilization of plasmid vectors by the partitioning function of broad-host-range plasmid RP4.

The genes for biosynthesis of the biodegradable polymer poly-beta-hydroxybutyric acid (PHB) cloned from Alcaligenes eutrophus H16 were used for synthesis of PHB with recombinant Escherichia coli strains. It was recognized that the PHB-biosynthesis genes cause segregational instability to the plasmids used as vectors. Recombinant PHB-plasmids are rapidly lost from host cells and plasmid-free cells occur at high rates, even under conditions of selection for the plasmids. Cloning the partitioning region of plasmid RP4 onto such plasmids resulted in a high degree of stabilization. These par-stabilized recombinant PHB-plasmids could be maintained quite efficiently in batch cultivation experiments in the absence of any selection pressure.

Molecular characterization and functional analysis in Aspergillus nidulans of the 5'-region of the Penicillium chrysogenum isopenicillin N synthetase gene.

The isopenicillin N synthetase gene (pcbC) was isolated from a genomic library of Penicillium chrysogenum BC39813, a penicillin production strain. The nucleotide sequence, including 555 bp upstream of the translation start site was determined. Various deletions within the pcbC 5'-region were constructed and linked to the Escherichia coli lacZ gene. An Aspergillus nidulans argB strain was transformed with DNA of these constructions. The region essential for promoter function could be localized between positions -307 and -89 by analyzing beta-galactosidase expression of transformants containing a single copy of the corresponding plasmid integrated at the homologous argB locus. A region responsible for regulatory effects concerning nitrogen metabolism was identified by determining beta-galactosidase activities in cell-lysates of transformants cultivated under varying growth conditions. Two major transcription start sites at positions -131 and -132, as well as a further upstream located site at position -397 +/- 1 could be located by primer extension studies employing RNA isolated from P. chrysogenum BC39813.

Cloning and characterization of the gene for the thermostable xylanase XynA from Thermomyces lanuginosus.

A thermostable xylanase from the filamentous fungus Thermomyces lanuginosus (DSM 5826) was purified. This enzyme has an apparent molecular weight of 24-26 kDa as determined by SDS polyacrylamide gel electrophoresis. cDNA and genomic DNA fragments coding for this enzyme were cloned and sequenced. The cDNA contains an open reading frame encoding a polypeptide of 225 amino acids and was functionally expressed in E. coli as a LacZ fusion protein. Comparison of the cDNA sequence with the genomic DNA sequence showed that the xylanase was encoded by two exons interrupted by an intron of 106 bp. Comparison of the deduced amino acid sequence to other published xylanases revealed high homology to xylanases of the family G glycanases.

Detection of a new enzyme for stereoselective hydrolysis of linalyl acetate using simple plate assays for the characterization of cloned esterases from Burkholderia gladioli.

Plate assays were developed for the identification of specific hydrolytic activities of esterases from Burkholderia gladioli, cloned and expressed in Escherichia coli. Clones showing different substrate specificities were identified by fluorescence or azo-dye formation caused by the released alcohol moiety of the hydrolyzed substrates, or by colour change of pH indicators mediated by decreased pH. The use of 1-hydroxypyrene-3,6,8-trisulfonic acid (HPTS-) esters and linalyl acetate for these assays clearly allowed to discriminate substrate specificities for two different cloned esterases, EP6 and EP10. Long chain fatty acid HPTS-esters were only hydrolyzed by the EP10 clone. On the other hand, the EP6 clone showed significant activity in hydrolysis of the sterically hindered ester linalyl-acetate. Enantioselective hydrolysis of linalyl acetate could be verified with a crude EP6 preparation on a preparative scale.

Tetronic acid derivatives from Aspergillus panamensis. Production, isolation, characterization and biological activity.

When Aspergillus panamensis, CBS 120.45, was grown on a medium with high carbon and low nitrogen content, it was found to produce six antibiotically active metabolites. All compounds were identified as tetronic acid derivatives; three of them were found to be identical with gregatins A, B, and D, previously reported as metabolites of Cephalosporium gregatum. The other three compounds are new. All six compounds show antibacterial activity towards Gram-negative and Gram-positive bacteria. Five of the antibiotics exhibit inhibitory effects on the macromolecular syntheses in cells of the ascitic form of EHRLICH carcinoma (ECA) of mice.