Effects of radiation damage and inelastic scattering on single-particle imaging of hydrated proteins with an X-ray Free-Electron Laser.

We present a computational case study of X-ray single-particle imaging of hydrated proteins on an example of 2-Nitrogenase-Iron protein covered with water layers of various thickness, using a start-to-end simulation platform and experimental parameters of the SPB/SFX instrument at the European X-ray Free-Electron Laser facility. The simulations identify an optimal thickness of the water layer at which the effective resolution for imaging the hydrated sample becomes significantly higher than for the non-hydrated sample. This effect is lost when the water layer becomes too thick. Even though the detailed results presented pertain to the specific sample studied, the trends which we identify should also hold in a general case. We expect these findings will guide future single-particle imaging experiments using hydrated proteins.

Quaternary Charge-Transfer Complex Enables Photoenzymatic Intermolecular Hydroalkylation of Olefins.

Intermolecular C-C bond-forming reactions are underdeveloped transformations in the field of biocatalysis. Here we report a photoenzymatic intermolecular hydroalkylation of olefins catalyzed by flavin-dependent 'ene'-reductases. Radical initiation occurs via photoexcitation of a rare high-order enzyme-templated charge-transfer complex that forms between an alkene, α-chloroamide, and flavin hydroquinone. This unique mechanism ensures that radical formation only occurs when both substrates are present within the protein active site. This active site can control the radical terminating hydrogen atom transfer, enabling the synthesis of enantioenriched γ-stereogenic amides. This work highlights the potential for photoenzymatic catalysis to enable new biocatalytic transformations via previously unknown electron transfer mechanisms.

An algal photoenzyme converts fatty acids to hydrocarbons.

Although many organisms capture or respond to sunlight, few enzymes are known to be driven by light. Among these are DNA photolyases and the photosynthetic reaction centers. Here, we show that the microalga Chlorella variabilis NC64A harbors a photoenzyme that acts in lipid metabolism. This enzyme belongs to an algae-specific clade of the glucose-methanol-choline oxidoreductase family and catalyzes the decarboxylation of free fatty acids to n-alkanes or -alkenes in response to blue light. Crystal structure of the protein reveals a fatty acid-binding site in a hydrophobic tunnel leading to the light-capturing flavin adenine dinucleotide (FAD) cofactor. The decarboxylation is initiated through electron abstraction from the fatty acid by the photoexcited FAD with a quantum yield >80%. This photoenzyme, which we name fatty acid photodecarboxylase, may be useful in light-driven, bio-based production of hydrocarbons.

Adaptation response of Pseudomonas fragi on refrigerated solid matrix to a moderate electric field.

BACKGROUND: Moderate electric field (MEF) technology is a promising food preservation strategy since it relies on physical properties-rather than chemical additives-to preserve solid cellular foods during storage. However, the effectiveness of long-term MEF exposure on the psychrotrophic microorganisms responsible for the food spoilage at cool temperatures remains unclear. RESULTS: The spoilage-associated psychrotroph Pseudomonas fragi MC16 was obtained from pork samples stored at 7 °C. Continuous MEF treatment attenuated growth and resulted in subsequent adaptation of M16 cultured on nutrient agar plates at 7 °C, compared to the control cultures, as determined by biomass analysis and plating procedures. Moreover, intracellular dehydrogenase activity and ATP levels also indicated an initial effect of MEF treatment followed by cellular recovery, and extracellular ß-galactosidase activity assays indicated no obvious changes in cell membrane permeability. Furthermore, microscopic observations using scanning and transmission electron microscopy revealed that MEF induced sublethal cellular injury during early treatment stages, but no notable changes in morphology or cytology on subsequent days. CONCLUSION: Our study provides direct evidence that psychrotrophic P. fragi MC16 cultured on nutrient agar plates at 7 °C are capable of adapting to MEF treatment.

Artificial Photosynthesis: Hybrid Systems.

Oxidoreductases are promising catalysts for organic synthesis. To sustain their catalytic cycles they require efficient supply with redox equivalents. Today classical biomimetic approaches utilizing natural electron supply chains prevail but artificial regeneration approaches bear the promise of simpler and more robust reaction schemes. Utilizing visible light can accelerate such artificial electron transport chains and even enable thermodynamically unfeasible reactions such as the use of water as reductant.This contribution critically summarizes the current state of the art in photoredoxbiocatalysis (i.e. light-driven biocatalytic oxidation and reduction reactions).

X-ray-induced catalytic active-site reduction of a multicopper oxidase: structural insights into the proton-relay mechanism and O2-reduction states.

During X-ray data collection from a multicopper oxidase (MCO) crystal, electrons and protons are mainly released into the system by the radiolysis of water molecules, leading to the X-ray-induced reduction of O2 to 2H2O at the trinuclear copper cluster (TNC) of the enzyme. In this work, 12 crystallographic structures of Thermus thermophilus HB27 multicopper oxidase (Tth-MCO) in holo, apo and Hg-bound forms and with different X-ray absorbed doses have been determined. In holo Tth-MCO structures with four Cu atoms, the proton-donor residue Glu451 involved in O2 reduction was found in a double conformation: Glu451a (∼7 Šfrom the TNC) and Glu451b (∼4.5 Šfrom the TNC). A positive peak of electron density above 3.5σ in an Fo - Fc map for Glu451a O(ℇ2) indicates the presence of a carboxyl functional group at the side chain, while its significant absence in Glu451b strongly suggests a carboxylate functional group. In contrast, for apo Tth-MCO and in Hg-bound structures neither the positive peak nor double conformations were observed. Together, these observations provide the first structural evidence for a proton-relay mechanism in the MCO family and also support previous studies indicating that Asp106 does not provide protons for this mechanism. In addition, eight composite structures (Tth-MCO-C1-8) with different X-ray-absorbed doses allowed the observation of different O2-reduction states, and a total depletion of T2Cu at doses higher than 0.2 MGy showed the high susceptibility of this Cu atom to radiation damage, highlighting the importance of taking radiation effects into account in biochemical interpretations of an MCO structure.

Enhancement of cell production in photosynthetic bacteria wastewater treatment by low-strength ultrasound.

The present study is aimed at enhanced cell production in together with pollution removal in photosynthetic bacteria wastewater treatment through low-strength ultrasound stimulation. The ultrasound strength was 0.3W/cm(2) with 40kHz frequency. Results showed 1-10min sonication significantly improved the cell production. The optimal sonication time was 2min. When the irradiation period was over 10min, a strong mechanical damage occurred. These phenomena could be explained by the changes of PSB dehydrogenase activity. Sonication stimulated the initial dehydrogenase activity; moderate sonication increased the cell activity while too-long sonication led to quick enzyme activity decrease. The optimal sonication strategy was 1min at the 0th hour and 1min at the 27th hour. The corresponding cell production and cell yield increased by 110% and 93% respectively, and the COD removal reached 98%. The cost of low-strength ultrasound was only 1% of the incremental sales value of cells.

New insights into the catalytic active-site structure of multicopper oxidases.

Structural models determined by X-ray crystallography play a central role in understanding the catalytic mechanism of enzymes. However, X-ray radiation generates hydrated electrons that can cause significant damage to the active sites of metalloenzymes. In the present study, crystal structures of the multicopper oxidases (MCOs) CueO from Escherichia coli and laccase from a metagenome were determined. Diffraction data were obtained from a single crystal under low to high X-ray dose conditions. At low levels of X-ray exposure, unambiguous electron density for an O atom was observed inside the trinuclear copper centre (TNC) in both MCOs. The gradual reduction of copper by hydrated electrons monitored by measurement of the Cu K-edge X-ray absorption spectra led to the disappearance of the electron density for the O atom. In addition, the size of the copper triangle was enlarged by a two-step shift in the location of the type III coppers owing to reduction. Further, binding of O2 to the TNC after its full reduction was observed in the case of the laccase. Based on these novel structural findings, the diverse resting structures of the MCOs and their four-electron O2-reduction process are discussed.

Molecular characterization and primary functional analysis of PeVDE, a violaxanthin de-epoxidase gene from bamboo (Phyllostachys edulis).

KEY MESSAGE: PeVDE was expressed primarily in bamboo leaves, which was up-regulated under high light. The protein encoded by PeVDE had enzyme activity of catalyzing violaxanthin (V) to zeaxanthin (Z) through antheraxanthin (A) as assay shown in vitro. Violaxanthin de-epoxidase (VDE), a key enzyme of xanthophyll cycle, catalyzes conversion from violaxanthin (V) to zeaxanthin (Z) through antheraxanthin (A) to protect photosynthesis apparatus. A cDNA, PeVDE, encoding a VDE was isolated from bamboo (Phyllostachys edulis) by RT-PCR and RACE methods. PeVDE is 1,723 bp and contains an ORF encoding 451 amino acids, with a transit peptide of 103 amino acids. The mature protein is deduced to have 348 amino acids with a calculated molecular weight of 39.6 kDa and a theoretic isoelectric point of 4.5. Semi-quantitative RT-PCR assay indicated that the highest expression level of PeVDE was in leaf, which agreed with the accumulation pattern of PeVDE protein. Real time PCR results showed that PeVDE was up-regulated and reached the highest level after the treatment (1,200 µmo1 m(-2) s(-1)) for 2 h, then decreased and kept at the level similar to that of 0.5 h after treatment for 8 h. To investigate the function of PeVDE, mature protein was heterologously expressed in Escherichia coli and the enzymatic activity assay was carried out using V as substrate. The pigments that formed in the reaction mixture were extracted and analyzed by HPLC method. Besides V, A and Z were detected in the reaction mixture, which indicated that the recombinant protein exhibited enzymatic activity of catalyzing V into Z through A. This study indicates that PeVDE functions through regulating the components of xanthophyll cycle, which might be one of the critical factors that contribute to the growth of bamboo under naturally varying light conditions.

Cloning and characterization of a UV-B-inducible maize flavonol synthase.

Flavonols are important compounds for conditional male fertility in maize (Zea mays) and other crops, and they also contribute to protecting plants from UV-B radiation. However, little continues to be known on how maize and other grasses synthesize flavonols, and how flavonol biosynthesis is regulated. By homology with an Arabidopsis flavonol synthase (AtFLS1), we cloned a maize gene encoding a protein (ZmFLS1) capable of converting the dihydrokaempferol (DHK) and dihydroquercetin (DHQ) dihydroflavonols to the corresponding flavonols, kaempferol (K) and quercetin (Q). Moreover, ZmFLS1 partially complements the flavonol deficiency of the Arabidopsis fls1 mutant, and restores anthocyanin accumulation to normal levels. We demonstrate that ZmFLS1 is under the control of the anthocyanin (C1/PL1 + R/B) and 3-deoxy flavonoid (P1) transcriptional regulators. Indeed, using chromatin immunoprecipitation (ChIP) experiments, we establish that ZmFLS1 is an immediate direct target of the P1 and C1/R regulatory complexes, revealing similar control as for earlier steps in the maize flavonoid pathway. Highlighting the importance of flavonols in UV-B protection, we also show that ZmFLS1 is induced in maize seedlings by UV-B, and that this induction is in part mediated by the increased expression of the P1, B and PL1 regulators. Together, our results identify a key flavonoid biosynthetic enzyme so far missed in maize and other monocots, and illustrate mechanisms by which flavonol accumulation is controlled in maize.

Inactivation of chosen dehydrogenases by the products of water radiolysis and secondary albumin and haemoglobin radicals.

PURPOSE: Inactivation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), alcohol dehydrogenase (ADH) and lactate dehydrogenase (LDH) by products of water radiolysis and by secondary radicals localized on haemoglobin (Hb) and human albumin (HSA) was studied. MATERIALS AND METHODS: Aqueous solutions of ADH, GAPDH and LDH were irradiated under air and under nitrous oxide (N2O) in the absence and in the presence of Hb or HSA. In order to determine the effectiveness of inactivation of the enzymes by radicals localized on Hb and HSA, the inactivation efficiency determined experimentally was compared with that calculated under assumption that only hydroxyl radicals are responsible for the enzyme inactivation. RESULTS: In the absence of other proteins, under air, GAPDH showed the highest radiation sensitivity, followed by ADH and LDH. The sequence was reverse under anaerobic atmosphere. Oxygen increased considerably the inactivation of GAPDH and ADH. Secondary albumin and haemoglobin radicals brought about considerable inactivation of GAPGH and ADH. Albumin radicals (HSA) generated under N2O inactivated GAPDH and ADH more effectively than haemoglobin radicals (Hb). Under air, however, inactivation of GAPDH and ADH by haemoglobin peroxyl radicals was higher than by albumin peroxyl radicals. LDH was resistant to inactivation by haemoglobin and albumin radicals, and peroxides of these proteins. CONCLUSIONS: In the light of these results and literature data, the observed differences in the effectiveness of inactivation of the dehydrogenases studied by secondary protein radicals depend on the amino acid residues present at the active site and in its close neighborhood and on the number of amino acid residues available on the protein surface.

Antitumor effects of a natural anthracycline analog (Aloin) involve altered activity of antioxidant enzymes in HeLaS3 cells.

The antiproliferative and cytotoxic potential of the natural anthracycline aloin from Aloe vera was tested on human uterine carcinoma HeLaS3 cells. Aloin showed a pronounced antiproliferative effect at physiological concentration (IC50 = 97 microM), caused cell cycle arrest in the S phase and markedly increased HeLaS3 cell apoptosis (to 24%). In the concentration range of 20-100 microM, its action was accompanied by remarkable changes in the activity of almost all antioxidant enzymes: MnSOD activity was increased many fold, while CuZnSOD and iNOS activities were inhibited. Moreover, inhibition of CuZnSOD was shown to occur by direct aloin interaction with the enzyme. As catalase activity was not changed, it is suggested that such conditions were responsible for antiproliferative and cytotoxic effects owing to accumulation of H2O2. Aloin alone was a more potent proapoptotic agent than a 2 Gy fractional dose of ionizing radiation or a combination of the two. Compared to other currently used therapeutics, aloin, due to its less undesirable side effects and antimetastatic potential, may prove to be the agent of choice on which clinical protocols for the treatment of human cervical carcinoma should rely in future.

[Correction of free-radical processes in patients with chronic brain ischemia with intravenous laser irradiation of blood].

We examined lipid peroxidation (LPO) in 130 patients with chronic brain ischemia (CBI). Primary and secondary LPO products, oxidative and antioxidative enzymes were studied in the course of therapy with antioxidant drug cytoflavin and intravenous laser radiation of blood (ILRB). The latter proved to have a normalizing action on LPO and antioxidant defense. With progression of CBI, effects of ILRB on enzymatic activity weakens due to depletion of endogenic antioxidants. This necessitates administration of exogenic antioxidants (cytoflavin) for complex correction of free radical processes. This conclusion allows recommending combined schemes of therapy for patients with CBI stage II and III.

[The restoration of the enzyme activity of chernozem soil after gamma-irradiation].

The Influence of gamma-radiation by dozes 1, 5, 10 and 20 kGy on enzyme activity of ordinary chemozem were studied. Dynamics of the restoration of the enzyme activity after the influence of gamma-radiation in model experiments in 3, 30, 90 and 180 days was investigated. The doze 1 kGy did no statistically significant influence on the investigated enzymes. Dehydrogenase is more radiosensitive enzyme than catalase. Values of the saccharase activity differed a significant variation so in most cases it has not been registered statistically significant changes. In 90-180 days of the incubation enzymes activity was restored up to control values. Dehydrogenase activity in 180 days in variants with dozes 10 and 20 kGy was restored up to a level of the control, over variants with dozes 1 and 5 kGy--is higher than the control over 78% and 23% accordingly. Saccharase activity in 180 days after the influence of gamma-radiation with a doze 20 kGy was on 61% lower than the control.

FRA16D common chromosomal fragile site oxido-reductase (FOR/WWOX) protects against the effects of ionizing radiation in Drosophila.

Fragile sites are chromosomal structures that have been proposed to have a determining role in cancer-associated DNA instability. The human WWOX gene spans the FRA16D chromosomal fragile site, the common minimal region of homozygous deletion found in adenocarcinomas and three out of five translocation breakpoints in multiple myeloma. Transcripts from the alternatively spliced WWOX gene encode proteins with common N-terminal WW domains and variable homology to the oxidoreductase family of proteins. In this study, the Drosophila orthologue of the WWOX gene was identified and subjected to mutagenesis via homologous recombination. The resultant DmWWOX1 mutants were viable but exhibited an increased sensitivity to ionizing radiation. This radiation sensitivity was rescued by reintroduction and expression of either the wild-type Drosophila or human WWOX genes. Thus, the protective function of DmWWOX in response to irradiation in Drosophila is conserved with human WWOX (hWWOX). This is consistent with a protective role for hWWOX where aberrant expression, as a result of breakage at the associated fragile site, could contribute directly to cancer progression.

Construction of an electro-enzymatic bioreactor for the production of (R)-mandelate from benzoylformate.

Coupling both the electrocatalytic recycling of NADH and the enzymatic reduction of the substrate was used to produce (R)-mandelate from benzoylformate using benzoylformate reductase (BFR). The reduction of benzoylformate by BFR in combination with FAD-mediated electrolysis (at -0.5 V vs. Ag/AgCl) was complete in about 18 h and gave 47.5 mM (R)-mandelate from 50 mM substrate, while the process involving MV2+-mediated procedure (at -0.7 V vs. Ag/AgCl) produced 40 mM (R)-mandelate after 30 h. The overpotential for the NAD+ reduction could be decreased by about 0.2 V by substituting a toxic viologen derivative, MV2+, with a natural electron carrier, FAD. MV2+, however, decreased the productivity as BFR lost about 50% of its initial activity after 6 d in its presence.

Supplementary ultraviolet-B radiation induces a rapid reversal of the diadinoxanthin cycle in the strong light-exposed diatom Phaeodactylum tricornutum.

A treatment of the diatom Phaeodactylum tricornutum with high light (HL) in the visible range led to the conversion of diadinoxanthin (Dd) to diatoxanthin (Dt). In a following treatment with HL plus supplementary ultraviolet (UV)-B, the Dt was rapidly epoxidized to Dd. Photosynthesis of the cells was inhibited under HL + UV-B. This is accounted for by direct damage by UV-B and damage because of the UV-B-induced reversal of the Dd cycle and the associated loss of photoprotection. The reversal of the Dd cycle by UV-B was faster in the presence of dithiothreitol, an inhibitor of the Dd de-epoxidase. Our results imply that the reversal of the Dd cycle by HL + UV-B was caused by an increase in the rate of gross Dt epoxidation, whereas the de-epoxidation of Dd was unaffected by UV-B. This is further supported by our finding that the in vitro de-epoxidation activity and the affinity toward the cosubstrate ascorbic acid of the Dd de-epoxidase were both unaffected by UV-B pretreatment of intact cells. We provide evidence that Dt epoxidation is normally down-regulated by a high pH gradient under HL. It is proposed that supplementary UV-B affected the pH gradient across the thylakoid membrane, which disrupted the down-regulation of Dt epoxidation and led to the observed increase in the rate of Dt epoxidation.

Effects of low chronic doses of ionizing radiation on antioxidant enzymes and G6PDH activities in Stipa capillata (Poaceae).

Stipa capillata (Poaceae) seeds were harvested from a control area (displaying a gamma dose rate of 0.23 micro Sv h(-1)) (C plants) and from two contaminated areas (5.4 and 25 micro Sv h(-1)) on the Semipalatinsk nuclear test site (SNTS) in Kazakhstan. The plants were grown for 124 d in a greenhouse under controlled conditions and exposed to three different treatments: (0) control; (E) external gamma irradiation delivered by a sealed 137Cs source with a dose rate of 66 micro Sv h(-1); (E+I) E treatment combined with internal beta irradiation due to contamination by 134Cs and 85Sr via root uptake from the soil. The root uptake led to a contamination of 100 Bq g(-1) for 85Sr and 5 Bq g(-1) for 134Cs (of plant dry weight) as measured at harvest. The activity of SOD, APX, GR, POD, CAT, G6PDH, and MDHAR enzymes was measured in leaves. Under (0) treatment, all enzymes showed similar activities, except POD, which had higher activity in plants originating from contaminated areas. Treatment (E) induced an enhancement of POD, CAT, GR, SOD, and G6PDH activities in plants originating from contaminated areas. Only control plants showed any stimulation of APX activity. Treatment (E+I) had no significant effect on APX, GR, CAT, and POD activities, but MDHAR activity was significantly reduced while SOD and G6PDH activities were significantly increased. The increase occurred in plants from all origins for SOD, with a greater magnitude as a function of their origin, and it occurred only in plants from the more contaminated populations for G6PDH. This suggests that exposure to a low dose rate of ionizing radiation for almost a half century in the original environment of Stipa has led to natural selection of the most adapted genotypes characterized by an efficient induction of anti-oxidant enzyme activities, especially SOD and G6PDH, involved in plant protection against reactive oxygen species.