Relevance of the flavin binding to the stability and folding of engineered cholesterol oxidase containing noncovalently bound FAD

The flavoprotein cholesterol oxidase (CO) from Brevibacterium sterolicum is a monomeric flavoenzyme containing one molecule of FAD cofactor covalently linked to His69. The elimination of the covalent link following the His69Ala substitution was demonstrated to result in a significant decrease in activity, in the midpoint redox potential of the flavin, and in stability with respect to the wild-type enzyme, but does not modify the overall structure of the enzyme. We used CO as a model system to dissect the changes due to the elimination of the covalent link between the flavin and the protein (by comparing the wild-type and H69A CO holoproteins) with those due to the elimination of the cofactor (by comparing the holo- and apoprotein forms of H69A CO). The apoprotein of H69A CO lacks the characteristic tertiary structure of the holoprotein and displays larger hydrophobic surfaces; its urea-induced unfolding does not occur by a simple two-state mechanism and is largely nonreversible. Minor alterations in the flavin binding region are evident between the native and the refolded proteins, and are likely responsible for the low refolding yield observed. A model for the equilibrium unfolding of H69A CO that also takes into consideration the effects of cofactor binding and dissociation, and thus may be of general significance in terms of the relationships between cofactor uptake and folding in flavoproteins, is presented.     

Proteomics of rat hypothalamus,hippocampus and pre-frontal/frontal cortex after central administration of the neuropeptide PACAP

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide that exerts pleiotropic functions, acting as a hypophysiotropic factor, a neurotrophic and a neuroprotective agent. The molecular pathways activated by PACAP to exert its physiological roles in brain are incompletely understood. In this study, adrenocorticotropic hormone (ACTH), prolactin, luteinising hormone (LH), follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), brain-derived neurotrophic factor and corticosterone blood levels were determined before and 20, 40, 60, and 120 min after PACAP intracerebroventricular administration. PACAP treatment increased ACTH, corticosterone, LH and FSH blood concentrations, while it decreased TSH levels. A proteomics investigation was carried out in hypothalamus, hippocampus and pre-frontal/frontal cortex (P/FC) using 2-dimensional gel electrophoresis at 120 min, the end-point suggested by studies on PACAP hypophysiotropic activities. Spots showing statistically significant alterations after PACAP treatment were identified by Matrix-assisted laser desorption/ionization-Time of flight mass spectrometry. Identified proteins were consistent with PACAP involvement in different molecular processes in brain. Altered expression levels were observed for proteins involved in cytoskeleton modulation and synaptic plasticity: actin in the hypothalamus; stathmin, dynamin, profilin and cofilin in hippocampus; synapsin in P/FC. Proteins involved in cellular differentiation were also modulated: glutathione-S-transferase α and peroxiredoxin in hippocampus; nucleoside diphosphate kinase in P/FC. Alterations were detected in proteins involved in neuroprotection, neurodegeneration and apoptosis: ubiquitin carboxyl-terminal hydrolase isozyme L1 and heat shock protein 90-β in hypothalamus; α-synuclein in hippocampus; glyceraldehyde-3-phosphate dehydrogenase and prohibitin in P/FC. This proteomics study identified new proteins involved in molecular mechanisms mediating PACAP functions in the central nervous system.     

Unfolding intermediate in the peroxisomal flavoprotein D-amino acid oxidase

The flavoenzyme d-amino acid oxidase (DAAO) from Rhodotorula gracilis is a peroxisomal enzyme and a prototypical member of the glutathione reductase family of flavoproteins. DAAO is a stable homodimer with a FAD molecule tightly bound to each 40-kDa subunit. In this work, the urea-induced unfolding of dimeric DAAO was compared with that of a monomeric form of the same protein, a deleted dimerization loop mutant. By using circular dichroism spectroscopy, protein and flavin fluorescence, 1,8-anilinonaphtalene sulfonic acid binding and activity assays, we demonstrated that the urea-induced unfolding of DAAO is a three-state process, yielding an intermediate, and that this process is reversible. The intermediate species lacks the catalytic activity and the characteristic tertiary structure of native DAAO but has significant secondary structure and retains flavin binding. Unfolding of DAAO proceeds through formation of an expanded, partially unfolded inactive intermediate, characterized by low solubility, by increased exposure of hydrophobic surfaces, and by increased sensitivity to trypsin of the beta-strand F5 belonging to the FAD binding domain. The oligomeric state does not modify the inferred folding process. The strand F5 is in contact with the C-terminal alpha-helix containing the Ser-Lys-Leu sequence corresponding to the type 1 peroxisomal targeting signal, and this structural element interacts with the N-terminal betaalphabeta flavin binding motif (Rossmann fold). The expanded conformation of the folding intermediate (and in particular the higher disorder of the mentioned secondary structure elements) could match the structure of the inactive holoenzyme required for in vivo trafficking of DAAO through the peroxisomal membrane.     

Baculovirus-resistant Anticarsia gemmatalis responds differently to dietary rutin

The velvetbean caterpillar, Anticarsia gemmatalis (Hübner) (Lepidoptera: Noctuidae), the major soybean [Glycine max (L.) Merrill (Fabaceae)] defoliator pest in Brazil can be controlled by a specific and virulent nuclear polyhedrosis virus (AgMNPV). Flavonoids such as rutin (quercetin 3‐O‐rhamnosylglucoside) were identified in soybean; it is known that this compound plays an important role in plant defense against lepidopteran pests. Studies were carried out to evaluate the biological and physiological activity of rutin (0.65 and 1.30%) on populations of A. gemmatalis resistant and susceptible to AgMNPV. Larvae from the resistant population were more negatively influenced by rutin, in comparison to larvae of the susceptible population, even with the addition of the lowest level of the flavonoid (0.65%) to the insect diet. The highest mortality (98%) was observed in the resistant population, when larvae fed on the diet containing 1.30% of rutin. Elongation of the feeding time, smaller initial larval weight, and pupal weight was observed on the virus‐resistant and ‐susceptible populations after adding 0.65 and 1.30% rutin to the diet. Larvae of the resistant population to AgMNPV fed on diet plus rutin 0.65% were also less efficient in the conversion of ingested and digested food into biomass.     

Dissection of the structural determinants involved in formation of the dimeric form of D-amino acid oxidase from Rhodotorula gracilis: role of the size of the betaF5-betaF6 loop

The role of the long loop connecting beta-strands F5 and F6 (21 amino acids, Pro302-Leu-Asp-Arg-Thr-Lys-Ser-Pro-Leu-Ser-Leu-Gly-Arg-Gly-Ser-Ala-Arg-Ala-Ala-Lys-Glu322) present in Rhodotorula gracilis d-amino acid oxidase (RgDAAO) was investigated by site-directed mutagenesis. This loop was proposed to play an important role in the 'head-to-tail' monomer-monomer interaction of this dimeric flavoenzyme: in particular, by means of electrostatic interactions between positively charged residues of the betaF5-betaF6 loop of one monomer and negatively charged residues belonging to the alpha-helices I3' and I3" of the other monomer. We produced a mutant of RgDAAO (namely, DAAO-DeltaLOOP2), in which only minor structural perturbations were introduced (only five amino acids were deleted; new sequence of the betaF5-betaF6 loop is Pro302-Leu-Asp-Arg-Thr-Leu-Gly-Arg-Gly-Ser-Ala-Arg-Ala-Ala-Lys-Glu317), and the charge of the betaF5-betaF6 loop not modified. The DeltaLOOP2 mutant is monomeric, has a weaker binding with the FAD cofactor, a decrease of the kinetic efficiency, and slight modifications in its spectral properties. The short version of the loop does not allow a correct monomer-monomer interaction, and its presence in the monomeric DAAO is a destabilizing structural element since the DeltaLOOP2 mutant is highly susceptible to proteolysis. These results, confirming the role of this loop in the subunits interaction and thus in stabilization of the sole dimeric form of RgDAAO, put forward the evidence that even a short deletion of the loop generates a consistent variation of the enzyme structure-function properties.     

On the mechanism of Rhodotorula gracilis D-amino acid oxidase: role of the active site serine 335

Serine 335 at the active site of D-amino acid oxidase from the yeast Rhodotorula gracilis (RgDAAO) is not conserved in other DAAO sequences. To assess its role in catalysis, it was mutated to Gly, the residue present in mammalian DAAO, an enzyme with a 35-fold lower turnover number with D-alanine. The spectral and ligand binding properties of the S335G mutant are similar to those of wild-type enzyme, suggesting an active site with minimally altered electrostatic properties. The S335G mutant is catalytically active, excluding an essential role of S335 in catalysis. However, S335-OH contributes to the high efficiency of the mutant enzyme since the catalytic activity of the latter is lower due to a decreased rate of flavin reduction relative to wild-type RgDAAO. Catalytic rates are pH-dependent and appear to converge to very low, but finite and similar values at low pH for both wild-type and S335G RgDAAO. While this dependence exhibits two apparent pKs with wild-type RgDAAO, with the S335G mutant a single, apparent pK approximately 8 is observed, which is attributed to the ionization of the alphaNH2 group of the bound substrate. Removal of S335-OH thus suppresses an apparent pK approximately 6. Both wild-type RgDAAO and the S335G mutant exhibit a substantial deuterium solvent kinetic isotope effect (> or =4) at pH<7 that disappears with increasing pH and reflects a pKapp=6.9 +/- 0.4. Interestingly, the substitution suppresses the activity towards d-lactate, suggesting a role of the serine 335 in removal of the substrate alpha-OH hydrogen.     

Production of recombinant cholesterol oxidase containing covalently bound FAD in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Cholesterol oxidase is an alcohol dehydrogenase/oxidase flavoprotein that catalyzes the dehydrogenation of C(3)-OH of cholesterol. It has two major biotechnological applications, i.e. in the determination of serum (and food) cholesterol levels and as biocatalyst providing valuable intermediates for industrial steroid drug production. Cholesterol oxidases of type I are those containing the FAD cofactor tightly but not covalently bound to the protein moiety, whereas type II members contain covalently bound FAD. This is the first report on the over-expression in Escherichia coli of type II cholesterol oxidase from Brevibacterium sterolicum (BCO).     

Strong seasonal disequilibrium measured between the oxygen isotope signals of leaf and soil CO2 exchange

The oxygen isotope composition (δ¹⁸O) of atmospheric CO₂ is among a very limited number of tools available to constrain estimates of the biospheric gross CO₂ fluxes, photosynthesis and respiration at large scales. However, the accuracy of the partitioning strongly depends on the extent of isotopic disequilibrium between the signals carried by these two gross fluxes. Chamber‐based field measurements of total CO₂ and CO¹⁸O fluxes from foliage and soil can help evaluate and refine our models of isotopic fractionation by plants and soils and validate the extent and pattern of isotopic disequilibrium within terrestrial ecosystems. Owing to sampling limitations in the past, such measurements have been very rare and covered only a few days. In this study, we coupled automated branch and soil chambers with tuneable diode laser absorption spectroscopy techniques to continuously capture the δ¹⁸O signals of foliage and soil CO₂ exchange in a Pinus pinaster Aït forest in France. Over the growing season, we observed a seasonally persistent isotopic disequilibrium between the δ¹⁸O signatures of net CO₂ fluxes from leaves and soils, except during rain events when the isotopic imbalance became temporarily weaker. Variations in the δ¹⁸O of CO₂ exchanged between leaves, soil and the atmosphere were well explained by theory describing changes in the oxygen isotope composition of ecosystem water pools in response to changes in leaf transpiration and soil evaporation.     

Termite cohabitation: the relative effect of biotic and abiotic factors on mound biodiversity

1. Termites are important ecosystem engineers that improve primary productivity in trees and animal diversity outside their mounds. However, their ecological relationship with the species nesting inside their mounds is poorly understood. 2. The presence of termite cohabitant colonies inside 145 Cornitermes cumulans mounds of known size and location was recorded. Using network‐theoretical methods in conjunction with a suite of statistical analyses, the relative influence of biotic and abiotic drivers of termite within‐mound diversity on the composition and species richness of the termite community was investigated, specifically builder presence and physical aspects of the mound. 3. We found that richness inside the mound increases with mound size, and the species similarity between mounds decreases with distance. The physical attributes (abiotic drivers) of termite mounds (size and relative distance to other mounds) are the strongest predictors of termite species richness and composition. The biotic driver (presence of a builder colony) has an important, though smaller, negative effect on within‐mound termite species richness. 4. The findings suggest that the termites' physical manipulation of their environment is an important driver of within‐mound community diversity. More generally, the approach taken here, using a combination of statistical and network‐theoretical methods, can be used to determine the relative importance of abiotic and biotic drivers of diversity in a wide range of communities of interacting species.     

Maternal LAMP/p55gagHIV-1 DNA immunization induces in utero priming and a long-lasting immune response in vaccinated neonates

Infants born to HIV-infected mothers are at high risk of becoming infected during gestation or the breastfeeding period. A search is thus warranted for vaccine formulations that will prevent mother-to-child HIV transmission. The LAMP/gag DNA chimeric vaccine encodes the HIV-1 p55gag fused to the lysosome-associated membrane protein-1 (LAMP-1) and has been shown to enhance anti-Gag antibody (Ab) and cellular immune responses in adult and neonatal mice; such a vaccine represents a new concept in antigen presentation. In this study, we evaluated the effect of LAMP/gag DNA immunization on neonates either before conception or during pregnancy. LAMP/gag immunization of BALB/c mice before conception by the intradermal route led to the transfer of anti-Gag IgG1 Ab through the placenta and via breastfeeding. Furthermore, there were an increased percentage of CD4+CD25+Foxp3+T cells in the spleens of neonates. When offspring were immunized with LAMP/gag DNA, the anti-Gag Ab response and the Gag-specific IFN-γ-secreting cells were decreased. Inhibition of anti-Gag Ab production and cellular responses were not observed six months after immunization, indicating that maternal immunization did not interfere with the long-lasting memory response in offspring. Injection of purified IgG in conjunction with LAMP/gag DNA immunization decreased humoral and cytotoxic T-cell responses. LAMP/gag DNA immunization by intradermal injection prior to conception promoted the transfer of Ab, leading to a diminished response to Gag without interfering with the development of anti-Gag T- and B-cell memory. Finally, we assessed responses after one intravenous injection of LAMP/gag DNA during the last five days of pregnancy. The intravenous injection led to in utero immunization. In conclusion, DNA vaccine enconding LAMP-1 with Gag and other HIV-1 antigens should be considered in the development of a protective vaccine for the maternal/fetal and newborn periods.