Alternative oxidation by isopenicillin N synthase observed by X-ray diffraction

Background: Isopenicillin N synthase (IPNS) catalyses formation of bicyclic isopenicillin N, precursor to all penicillin and cephalosporin antibiotics, from the linear tripeptide δ-( -α-aminoadipoyl)- -cysteinyl- -valine. IPNS is a non-haem iron(II)-dependent enzyme which utilises the full oxidising potential of molecular oxygen in catalysing the bicyclisation reaction. The reaction mechanism is believed to involve initial formation of the β-lactam ring (via a thioaldehyde intermediate) to give an iron(IV)-oxo species, which then mediates closure of the 5-membered thiazolidine ring.Results: Here we report experiments employing time-resolved crystallography to observe turnover of an isosteric substrate analogue designed to intercept the catalytic pathway at an early stage. Reaction in the crystalline enzyme–substrate complex was initiated by the application of high-pressure oxygen, and subsequent flash freezing allowed an oxygenated product to be trapped, bound at the iron centre. A mechanism for formation of the observed thiocarboxylate product is proposed.Conclusions: In the absence of its natural reaction partner (the N–H proton of the -cysteinyl- -valine amide bond), the proposed hydroperoxide intermediate appears to attack the putative thioaldehyde species directly. These results shed light on the events preceding β-lactam closure in the IPNS reaction cycle, and enhance our understanding of the mechanism for reaction of the enzyme with its natural substrate.     

Surface oxidation and reduction of small platinum particles observed by in-situ X-ray diffraction

A new method is presented, in which the constructive Bragg interference intensity of small metallic clusters during the formation or decay of a surface compound is measured by an in situ X-ray technique. First results for a series of platinum catalysts supported on silica gel show intensity losses on the 111-peak of up to 40% under oxygen following a standard pretreatment. These losses are restored reversibly under hydrogen. The average thickness of the disrupted surface layer ranges between 0.9 to 4.0 Å depending on the treatment temperature and platinum crystallite size.     

Design and synthesis of an isopenicillin N synthase mimic

Towards the aim of creating a functional mimic of isopenicillin N synthase, a small molecule designed to coordinate around iron(II) and model the enzyme active site has been prepared in nine synthetic steps from 2,6-bis(hydroxymethyl)pyridine, (S)-(+)-mandelic acid and pivaldehyde. One aspartate, two histidines and a water ligand in the natural enzyme are replaced by an α-hydroxy acid, pyridine and aniline in the model compound. Additionally, a free thiol designed to simulate the enzyme substrate, δ-(l-α-aminoadipoyl)-l-cysteinyl-d-valine, is linked to the ligand by a three carbon chain. We postulate that in the presence of molecular oxygen, the complex formed between this synthetic ligand and iron(II) will display oxidative chemistry similar to that observed in the active site of isopenicillin N synthase.     

Crystallographic studies on the reaction of isopenicillin N synthase with an unsaturated substrate analogue

Isopenicillin N synthase (IPNS) catalyses conversion of the linear tripeptide delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N (IPN), the central step in biosynthesis of the beta-lactam antibiotics. The unsaturated substrate analogue delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-vinylglycine (ACvG) has previously been incubated with IPNS and single product was isolated, a 2-alpha-hydroxymethyl isopenicillin N (HMPen), formed via a monooxygenase mode of reactivity. ACvG has now been crystallised with IPNS and the structure of the anaerobic IPNS:Fe(II):ACvG complex determined to 1.15 A resolution. Furthermore, by exposing the anaerobically grown crystals to high-pressure oxygen gas, a structure corresponding to the bicyclic product HMPen has been obtained at 1.60 A resolution. In light of these and other IPNS structures, and recent developments with related dioxygenases, the [2 + 2] cycloaddition mechanism for HMPen formation from ACvG has been revised, and a stepwise radical mechanism is proposed. This revised mechanism remains consistent with the observed stereospecificity of the transformation, but fits better with apparent constraints on the coordination geometry around the active site iron atom.     

N.M.R. and X-ray diffraction of the 7,7'-disodiumcromoglycate-water lyomesophases

Aqueous solutions of 7,7'-disodiumcromoglycate (7,7'-DSCG) form a variety of mesophases, whose natures depend on concentration and temperature. We have used optical microscopy, N.M.R. (of ²H, ¹⁷O and ²³Na) and X-ray techniques to construct a phase diagram for this system and to characterize the various mesophases. In the concentration range 3 to 25 wt % 7,7'-DSCG four different mesophases are found. On cooling the isotropic solution an opaque gel phase is first obtained, followed on further cooling by a birefringent glassy liquid. At concentrations below 14wt% it is nematic, while at higher concentrations it appears to be a different, more ordered, phase. At lower temperatures a hexagonal phase is formed which finally solidifies on cooling to - 15°C. The N.M.R. and X-ray characteristics of these mesophases are discussed.     

Non-isothermal kinetics of high temperature oxidation studied by a fast X-ray diffraction method

Temperature resolved X-ray diffraction using a diffractometer equipped with a high temperature device was applied to the study of the oxidation of Fe-ARMCO below 570°C. The oxidation products are identified in situ and the difference method yields curvesY(T) showing the global changes. These curves were calibrated to the microscopically measured end thickness of the oxide layer obtaining the growthx(T). A calculated curve including the Arrhenius relation was fitted to thex(T) curve yielding activation energy and values of the oxidation rate constant as a function of temperature.

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Zusammenfassung Unter Anwendung eines Diffraktometers mit einer Hochtemperatureinheit wurde unterhalb 570°C mittels Röntgendiffraktion die Oxidation von Fe-ARMCO untersucht. Die Oxidationsprodukte wurden in situ bestimmt und das Differenzverfahren ergabY(T)-Kurven, die die globalen Änderungen zeigen. Diese Kurven wurden anhand der mikroskopisch bestimmten resultierenden Dicken der Oxidschicht kalibriert, wobei man die Funktionx(T) erhält. Dieser Kurve wurde eine berechnete, die Arrheniussche Beziehung enthaltende Funktion angepaßt, wodurch man die Aktivierungsenergie und die Oxidations Geschwindigkeitskonstante als Funktion der Temperatur erhält.

     

Development of enzyme-linked immunosorbent assays for the detection of deacetoxycephalosporin C and isopenicillin N synthase activity

Although there are a number of existing assays for monitoring the activity of both isopenicillin N synthase (IPNS) and deacetoxycephalosporin C synthase (DAOCS), none have demonstrated the qualities required for screening a mutant library. Hence, enzyme-linked immunosorbent assays (ELISAs) for IPNS and DAOCS were developed based on the detection of the catalytic turnover products isopenicillin N and cephalexin/phenylacetyl-7-aminodeacetoxycephalosporanic acid (G-7-ADCA), respectively. These assays are relatively fast compared to existing assays, such as the hole-plate bioassay, and are amenable with high-throughput screening. Both the IPNS and DAOCS-ELISAs were optimised for use with crude protein extracts rather than purified protein, thereby eliminating any additional time required for purification. The ELISA developed for the detection of cephalexin had an IC₅₀ value of 154 ± 9 ng mL⁻¹ and LOD of 7.2 ± 2.2 ng mL⁻¹ under conditions required for the assay. Good recoveries and correlation was observed for spiked samples when the concentration of crude protein was kept below 1 mg mL⁻¹. The DAOCS-ELISA was found to have increased sensitivity compared to the hole-plate bioassay (10.3 μg mL⁻¹). The IPNS-ELISA did not significantly increase the sensitivity (approximately 5 μg mL⁻¹) compared to that of the hole-plate bioassay (16 μg mL⁻¹) for isopenicillin N. The minimum amount of crude protein extract required for producing detectable amounts of product for both assays was below 0.5% of the maximum amount of protein that the assay could contain without any effect on the ELISA. This suggests that when screening a mutant library, mutants producing low amounts of the product could still be detected using these assays.     

X-ray diffraction by liquid-crystalline elastomers

A new series of mesomorphic side chain polysiloxane networks has been recently synthesized in which the chemical nature of the linkage and the mesogenic group have been varied and the gelation conditions during the chemical reaction have been studied. This paper presents an X-ray diffraction study of the mesogenic group orientation in stretched samples of these networks. The angular extension of the so-called wide angle diffuse ring is used to estimate the orientational order of the mesogenic group versus strain. To perform these experiments, a special stretching device was developed and a new two-dimensional X-ray detector was used which allowed us to collect the data in a few minutes. On stretching, it was observed that the mesogenic groups orient themselves perpendicular to the stress direction for all of the samples but for one for which the parallel orientation prevailed. This prevents the establishment of a simple general law. From another point of view, the polymer concentration during the chemical reaction, which controls the gelation, is shown to be an important parameter with which to understand the physical properties: the networks synthesized below the gel point do not display reproducible and reversible behaviour, rather they flow when they are stretched. Conversely, all of the networks synthesized above the gel point really show the same well-defined behaviour independent of the sample history. Their orientational order increases regularly with the strain, first quickly, then moderately until it eventually saturates. This saturation value of the mesogenic group orientational order does not reach the nematic order parameter of the same (uncross-linked) mesomorphic side chain polymers. This suggests that the cross-links may create local tensions which disturb the nematic field.     

Invited Article. X-ray diffraction by mesomorphic comb-like polymers

This article presents a survey of the literature on X-ray diffraction by mesomorphic comb-like polymers. Special emphasis is placed upon two points: it is often possible to study the localization of the backbone in the smectic phases by considering the intensities of the Bragg reflections from the layers. It is also possible to observe different kinds of short range order and localized defects through their contribution to the X-ray diffuse scattering. For instance, the average S_A structure may be affected by layer undulations or disturbed by edge dislocations. By examining the many X-ray diffraction studies already published, it can be shown that the backbones have an ambiguous influence upon the molecular organization. They sometimes tend, for entropic reasons, to lessen the positional long range order or to create defects, whereas they sometimes promote short range order because they induce correlations among the mesogenic cores chemically linked to them.     

15N solid-NMR and X-ray diffraction studies of N-confused porphyrins

Using (15)N high-resolution solid-state NMR and X-ray diffraction, the structure of N-confused porphyrin (NCP) in the solid state was studied. A 1D (15)N magic angle spinning (MAS) experiment and a 2D dipolar assisted rotational resonance (DARR) (15)N-(15)N spin exchange experiment of N-confused tetratolylporphyrin (Tol) crystallized from CH(2)Cl(2)/hexane indicate that Tol is the inner 3H-type tautomer and has two magnetically different molecules in the unit cell. Further, a FSLG-2 & 4macr; 2 (1)H-(15)N dipolar recoupling NMR measurement indicates no fast ring flipping motion which is consistent with the planar structure in the X-ray analysis. The planarity of Tol is ascribed to crystal packing enforced by pi-pi stacking and CH-pi interactions.     

Protein environment facilitates O2 binding in non-heme iron enzyme. An insight from ONIOM calculations on isopenicillin N synthase (IPNS)

Binding of dioxygen to a non-heme enzyme has been modeled using the ONIOM combined quantum mechanical/molecular mechanical (QM/MM) method. For the present system, isopenicillin N synthase (IPNS), binding of dioxygen is stabilized by 8-10 kcal/mol for a QM:MM (B3LYP:Amber) protein model compared to a quantum mechanical model of the active site only. In the protein system, the free energy change of O2 binding is close to zero. Two major factors consistently stabilize O2 binding. The first effect, evaluated at the QM level, originates from a change in coordination geometry of the iron center. The active-site model artificially favors the deoxy state (O2 not bound) because it allows too-large rearrangements of the five-coordinate iron site. This error is corrected when the protein is included. The corresponding effect on binding energies is 3-6 kcal/mol, depending on the coordination mode of O2 (side-on or end-on). The second major factor that stabilizes O2 binding is van der Waals interactions between dioxygen and the surrounding enzyme. These interactions, 3-4 kcal/mol at the MM level, are neglected in models that include only the active site. Polarization of the active site by surrounding amino acids does not have a significant effect on the binding energy in the present system.     

Determination of nanoparticle sizes by X-ray diffraction

Different procedures for analysis of particle sizes by the X-ray diffraction method are compared by the example of nanoparticles of nickel and iron(3+) oxide (Fe₂O₃). A modified Warren-Averbach method is proposed for the analysis of the X-ray diffraction line profile based on the approximation by the Voigt function, which yields stable solutions, and the efficiency of the method is shown. The analysis within the frame-work of the Warren-Averbach method makes it possible to restore the distribution function of nanoparticles (crystallites) over true diameters, which satisfactorily correlates with electron microscopy data. The applicability of the Warren-Averbach method to the estimation of crystallite sizes by the analysis of a single diffraction line is substantiated. The range of the applicability of the Scherrer, Williamson-Hall, Warren-Averbach, and modified Warren-Averbach methods to the substructure analysis by the X-ray diffraction is determined as depending on the method of nanostructure formation.     

Recent progress in ultrafast X-ray diffraction.

X-ray diffraction with femtosecond time-resolution represents a direct probe of ultrafast structural changes in condensed matter. The generation of ultrashort X-ray pulses in laser-driven plasma and/or accelerator-based sources has made substantial progress, and has allowed for studies of transient structures with an unprecedented accuracy. Herein, recent work on transient crystalline structures is reviewed, with the focus on laser-based experiments.     

Uptake of liquid alcohols by the flexible Fe(III) metal-organic framework MIL-53 observed by time-resolved in situ X-ray diffraction

A comprehensive, time‐resolved, energy‐dispersive X‐ray diffraction study of the uptake of liquid alcohols (methanol, ethanol, propan‐1‐ol and propan‐2‐ol) by the flexible metal‐organic framework solid MIL‐53(Fe)[H₂O] is reported. In the case of the primary alcohols, a fluorinated version of the MIL‐53(Fe) host (C2/c symmetry V ca. 1000 ų), in which a fraction of framework hydroxides are replaced by fluoride, shows uptake of alcohols to give initially a partially expanded phase (C2/c symmetry, V ca. 1200 ų) followed by an expanded form of the material (either Imcm or Pnam symmetry, V ca. 1600 ų). In the case of methanol–water mixtures, the EDXRD data show that the partially open intermediate phase undergoes volume expansion during its existence, before switching to a fully open structure if concentrated methanol is used; analogous behaviour is seen if the initial guest is propan‐2‐ol, which then is replaced by pyridine, where a continuous shift of Bragg peaks within C2/c symmetry is observed. In contrast to the partially fluorinated materials, the purely hydroxylated host materials show little tendency to stabilise partially open forms of MIL‐53(Fe) with primary alcohols and the kinetics of guest introduction are markedly slower without the framework fluorination: this is exemplified by the exchange of water by propan‐2‐ol, where a partially open C2/c phase is formed in a step‐wise manner. Our study defines the various possible pathways of liquid‐phase uptake of molecular guests by flexible solid MIL‐53(Fe).     

X-ray diffraction method for the investigation of the oxidation kinetics in thin iron films

Summary The time-dependent decrease of the intensity of X-ray diffraction peaks due to the interaction of the investigated metals with gases can be measured in situ with the help of a recently developed ultrahigh-vaccum diffraction chamber. We have used this method to study the oxidation kinetics of about 450 nm thick iron films. The activation energy obtained from the temperature dependence of the kinetics at a relatively high oxygen exposure corresponds to the well-known cation vacancy diffusion while distinctly smaller values are found for short duration of exposure. This result leads to the conclusion that grain boundary diffusion becomes effective in the films at the early stages of the oxidation process.

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Röntgenographisches Verfahren zur Untersuchung der Oxidationskinetik in dünnen Eisenschichten

Zusammenfassung Die zeitliche Änderung der Intensität von Röntgenbeugungsreflexen, die von der Wechselwirkung der untersuchten Metalle mit Gasen herrührt, kann in situ mit Hilfe einer kürzlich entwickelten Ultrahochvakuum-Röntgenbeugungskammer gemessen werden. Wir haben diese Methode zum Studium der Oxidationskinetik ca. 450 nm dicker Eisenfilme herangezogen. Die Aktivierungsenergie, die man aus der Temperaturabhängigkeit der Kinetik bei relativ hohen Einwirkungszeiten des Sauerstoffs erhält, entspricht der wohlbekannten Kationenleerstellendiffusion, während deutlich kleinere Werte für kurze Einwirkungszeiten gefunden werden. Dieses Ergebnis legt den Schluß nahe, daß zu Beginn des Oxidationsprozesses eine Korngrenzendiffusion wirksam wird.

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Financial support of the Deutsche Forschungsgemeinschaft is gratefully acknowledged. Thanks are due to Mrs Schuster of the Institute of Material Science I of our University for preparing the SEM micrographs.     

X-ray diffraction by thermotropic main-chain polymers with side groups. I. rigid polyesters

Thermotropic poly(sulfonyl hydroquinone terephthalate) with either H or CH₃ in the lateral substituent was aligned by drawing from nematic melts. Virtually identical x-ray intensity distributions were recorded consisting of “disks” of intensity periodically spaced along the meridian and concentric rings of intensity lying in the equatorial plane of reciprocal space. They correspond to what is expected for the hexagonal packing of parallel periodic chains that are randomly displaced along the chain axes. Using interactive graphics, appropriate molecular models were deduced in conformity with the observed diffraction intensities.     

X-ray diffraction by thermotropic main-chain polymers with side groups. II. semiflexible polyesters

Semiflexible thermotropic aromatic polyesters with lateral groups were aligned magnetically and mechanically in monodomains after separation into high-molecular-weight (polymer) and low-molecular-weight (oligomer) fractions. Based on the x-ray intensity distributions recorded for a series of seven different substituents in the arylsulfonyl substituted group, a novel smectic structure was determined for H, F, Cl, OCH₃, and CH₃ in the para position while a normal nematic alignment prevailed when it contained Br or NO₂.