Structural analysis of flavinylation in vanillyl-alcohol oxidase

Vanillyl-alcohol oxidase (VAO) is member of a newly recognized flavoprotein family of structurally related oxidoreductases. The enzyme contains a covalently linked FAD cofactor. To study the mechanism of flavinylation we have created a design point mutation (His-61 --> Thr). In the mutant enzyme the covalent His-C8 alpha -flavin linkage is not formed, while the enzyme is still able to bind FAD and perform catalysis. The H61T mutant displays a similar affinity for FAD and ADP (K-d = 1.8 and 2.1 muM, respectively) but does not interact with FMN. H61T is about 10-fold less active with 4-(methoxymethyl)phenol) (k(cat) = 0.24 s(-1), K-m = 40 muM) than the wild-type enzyme. The crystal structures of both the hole and apo form of H61T are highly similar to the structure of wild-type VAO, indicating that binding of FAD to the apoprotein does not require major structural rearrangements. These results show that covalent flavinylation is an autocatalytical process in which His-BI plays a crucial role by activating His-422. Furthermore, our studies clearly demonstrate that in VAO, the FAD binds via a typical lock-and-key approach to a preorganized binding site

Covalent flavinylation is essential for efficient redox catalysis in vanillyl-alcohol oxidase

By mutating the target residue of covalent flavinylation in vanillyl-alcohol oxidase, the functional role of the histidyl-FAD bond was studied. Three His(422) mutants (H422A, H422T, and H422C) were purified, which all contained tightly but noncovalently bound FAD. Steady state kinetics revealed that the mutants have retained enzyme activity, although the turnover rates have decreased by 1 order of magnitude. Stopped-flow analysis showed that the H422A mutant is still able to form a stable binary complex of reduced enzyme and a quinone methide product intermediate, a crucial step during vanillyl-alcohol oxidase-mediated catalysis, The only significant change in the catalytic cycle of the H422A mutant is a marked decrease in reduction rate. Redox potentials of both wild type and H422A vanillyl-alcohol oxidase have been determined. During reduction of H422A, a large portion of the neutral flavin semiquinone is observed. Using suitable reference dyes, the redox potentials for the two one-electron couples have been determined: -17 and -113 mV. Reduction of wild type enzyme did not result in any formation of flavin semiquinone and revealed a remarkably high redox potential of +55 mV, The marked decrease in redox potential caused by the missing covalent histidyl-FAD bond is reflected in the reduced rate of substrate-mediated flavin reduction limiting the turnover rate. Elucidation of the crystal structure of the H422A mutant established that deletion of the histidyl-FAD bond did not result in any significant structural changes. These results clearly indicate that covalent interaction of the isoalloxazine ring with the protein moiety can markedly increase the redox potential of the flavin cofactor, thereby facilitating redox catalysis, Thus, formation of a histidyl-EAD bond in specific flavoenzymes might have evolved as a way to contribute to the enhancement of their oxidative power

Impact of the Volatile Cr-species' Attack on the Conductivity of La(Ni,Fe)O3

This study demonstrates the detrimental impact of Cr on the electronic conductivity of a $LaNi_{0.6}Fe_{0.4}O_3$ (LNF) porous cathode layer at 800 ºC. Vapor transport of Cr-species, originating from a porous metallic foam, and subsequent reaction with LNF results in a decrease of the electronic conductivity of the LNF-layer. Cr has been detected throughout the whole cross-section of the LNF-layer. Transmission electron microscopy revealed that Cr is gradually incorporated into the LNF-grains, while Ni is proportionally expelled. The progressing Cr deposition and penetration into the LNF-grains most likely explains the electronic conductivity drop. The Cr-poisoning impact on the electronic conductivity of the LNF porous layer is considerably smaller at 600ºC than at 800ºC. A tentative mechanism for the Cr attack and its influence on the electronic conductivity of the LNF layer will be presented

Managing the performance of maintenance personnel

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Trajectories of land use change in Europe: a model-based exploration of rural futures

Land use change is characterized by a high diversity of change trajectories depending on the local conditions, regional context and external influences. Policy intervention aims to counteract the negative consequences of these changes and provide incentives for positive developments. Region typologies are a common tool to cluster regions with similar characteristics and possibly similar policy needs. This paper provides a typology of land use change in Europe at a high spatial resolution based on a series of different scenarios of land use change for the period 2000-2030. A series of simulation models ranging from the global to the landscape level are used to translate scenario conditions in terms of demographic, economic and policy change into changes in European land use pattern. A typology developed based on these simulation results identifies the main trajectories of change across Europe: agricultural abandonment, agricultural expansion and urbanization. The results are combined with common typologies of landscape and rurality. The findings indicate that the typologies based on current landscape and ruralities are poor indicators of the land use dynamics simulated for the regions. It is advocated that typologies based on (simulated) future dynamics of land change are more appropriate to identify regions with potentially similar policy need

Ion-ion proton transfer reactions of bio-ions involving noncovalent interactions: Holomyoglobin

Multiply protonated horse skeletal muscle holomyoglobin and apomyoglobin have been subjected to ion-ion proton transfer reactions with anions derived from perfluoro-1,3dimethylcyclohexane in a quadrupole ion trap operated with helium as a bath gas at 1 mtorr. Neither the apomyoglobin nor holomyoglobin ions show any sign of fragmentation associated with charge state reduction to the 1 + charge state. This is particularly noteworthy for the holomyoglobin ions, which retain the noncovalently bound heme group. For example, no sign of heme loss is associated with charge state reduction from the 9 + charge state of holomyoglobin to the 1 + charge state despite the eight consecutive highly exothermic proton transfer reactions required to bring about this charge change. This result is consistent with calculations that show the combination of long ion lifetime and the high ion-helium collision rate relative to the ion-ion collision rate makes fragmentation unlikely for high mass ions in the ion trap environment even for noncovalently bound complexes of moderate binding strength. The ion-ion proton transfer rates for holo- and apomyoglobin ions of the same charge state also were observed to be indistinguishable, which supports the expectation that ion-ion proton transfer rates are insensitive to ion structure and are determined primarily by the attractive Coulomb field

Effect of dietary elaidic versus vaccenic acid on blood and liver lipids in the hamster

Male hamsters (30 per group) were fed five different semi-purified diets ad libitum. The diets, containing 30% of energy (en%) as fat, differed in their dietary fat composition (specified fatty acids exchanged at 10 en%) and were fed for 4 weeks. The five fatty acids compared in mixed triglycerides were elaidic acid (C18:1 9t), vaccenic acid (C18:1 11t), their cis-counterpart oleic acid (C18:1 9c), medium-chain fatty acids (MCFA; C8:0 and C10:0), and palmitic acid (C16:0). Compared with oleic acid, dietary MCFA and palmitic acid tended to increase blood cholesterol levels in the hamsters. The effect of elaidic and vaccenic acid on blood cholesterol did not differ from that of oleic acid. When elaidic acid and vaccenic acids were compared directly, the ratio of LDL/HDL-cholesterol in plasma was significantly higher in hamsters fed vaccenic acid than in those fed elaidic acid, and elaidic acid was incorporated at low levels, but more efficiently than vaccenic acid at the sn-2 position of platelet phospholipids. Biological consequences of this low incorporation are considered unlikely as levels of arachidonic acid (C20:4 n-6) and docosohexaenoic acid (C22:6 n-3) in the platelet phospholipids of all dietary groups did not differ. With respect to the effect on the LDL/HDL-cholesterol ratio, elaidic acid may be preferable to vaccenic acid. We conclude that this animal study does not provide evidence for the suggestion, based on epidemiological observations, that elaidic acid would be more detrimental to cardiovascular risk than vaccenic acid

Age of high-grade gneisses south of Grand Lake, Newfoundland

Crystalline rocks of the Steel Mountain Subzone of the Humber Zone in southwest Newfoundland give an age for granulite-grade metamorphism of 1498+9/-8 Ma, similar to ages from the Long Range inlier and northwestern Cape Breton Island. Peralkaline leucogranite was emplaced at 608 ± 4 Ma. The emplacement of anorthosite-gabbro complexes and amphibolite-grade metamorphism took place between these dates. The southern part of the Dunnage Zone (Central Gneiss Subzone), in contact with the Steel Mountain Subzone at the Long Range Fault, lacks Precambrian crystalline rocks, but was intruded by charnockitic plutons and metamorphosed to granulite facies at 460 ±10 Ma. This subzone was exhumed before 435 Ma. In the Meelpaeg Subzone of the Gander Zone, which is in contact with the Central Gneiss Subzone along the Victoria River Fault, the oldest intrusive component of a granoblastic migmatitic gneiss was emplaced at 418 ± 4 Ma. These data demonstrate that both the Long Range and Victoria River faults form major tectonic boundaries. Subzones appear to have been thrust westward in Silurian or later time. RÉSUMÉ Les roches cristallines de la sous-zone du mont Steel, dans la zone de Humber du sud-ouest de Terre-Neuve, ont donné des âges de 1498+9/-8 Ma pour le métamorphisme de haul grade, similaires à ceux de la boutonnière de Long Range et du nord de l'ile-du-Cap-Breton. Un leucogranite peralcalin s'est mis en place à 608 ± 4 Ma. L'intrusion des complexes à anorthosite-gabbro et le métamorphisme au faciès amphibolite se sont produits entre ces deux évènements. La partie sud de la zone de Dunnage (sous-zone de gneiss centrale), en contact avec la sous-zone du mont Steel a la faille de Long Range, ne contient pas de roches cristallines précambriennes mais à 616 recouped par des plutons charnockitiques et à 616 métamorphisée au faciès granulite à 460 ± 10 Ma. Cette sous-zone a été exhumge avant 435 Ma. Dans la sous-zone Mulpaeg de la zone de Gander, qui est mise en contact avec la sous-zone de gneiss centrale par la faille de la rivière Victoria, la phase intrusive la plus ancienne d'un gneiss migmatitique et granoblastique s'est mise en place à 418 ± 4 Ma. Ces données démontrent que les failles de Long Range et de la rivière Victoria sont des frontières tectoniques majeures. Les sous-zones semblent avoir subi un chevauchement vers l'ouest au plus tard au Silurien. [Traduit par le journal

A xylenol orange-based screening assay for the substrate specificity of flavin-dependent para-phenol oxidases

Vanillyl alcohol oxidase (VAO) and eugenol oxidase (EUGO) are flavin-dependent enzymes that catalyse the oxidation of para-substituted phenols. This makes them potentially interesting biocatalysts for the conversion of lignin-derived aromatic monomers to value-added compounds. To facilitate their biocatalytic exploitation, it is important to develop methods by which variants of the enzymes can be rapidly screened for increased activity towards substrates of interest. Here, we present the development of a screening assay for the substrate specificity of para-phenol oxidases based on the detection of hydrogen peroxide using the ferric-xylenol orange complex method. The assay was used to screen the activity of VAO and EUGO towards a set of twenty-four potential substrates. This led to the identification of 4-cyclopentylphenol as a new substrate of VAO and EUGO and 4-cyclohexylphenol as a new substrate of VAO. Screening of a small library of VAO and EUGO active-site variants for alterations in their substrate specificity led to the identification of a VAO variant (T457Q) with increased activity towards vanillyl alcohol (4-hydroxy-3-methoxybenzyl alcohol) and a EUGO variant (V436I) with increased activity towards chavicol (4-allylphenol) and 4-cyclopentylphenol. This assay provides a quick and efficient method to screen the substrate specificity of para-phenol oxidases, facilitating the enzyme engineering of known para-phenol oxidases and the evaluation of the substrate specificity of novel para-phenol oxidases