N-carboxyacyl and N-α-aminoacyl derivatives of aminoaldehydes as shared substrates of plant aldehyde dehydrogenases 10 and 7.

Aldehyde dehydrogenases (ALDHs) represent a superfamily of enzymes, which oxidize aldehydes to the corresponding acids. Certain families, namely ALDH9 and ALDH10, are best active with ω-aminoaldehydes arising from the metabolism of polyamines such as 3-aminopropionaldehyde and 4-aminobutyraldehyde. Plant ALDH10s show broad specificity and accept many different aldehydes (aliphatic, aromatic and heterocyclic) as substrates. This work involved the above-mentioned aminoaldehydes acylated with dicarboxylic acids, phenylalanine, and tyrosine. The resulting products were then examined with native ALDH10 from pea and recombinant ALDH7s from pea and maize. This investigation aimed to find a common efficient substrate for the two plant ALDH families. One of the best natural substrates of ALDH7s is aminoadipic semialdehyde carrying a carboxylic group opposite the aldehyde group. The substrate properties of the new compounds were demonstrated by mass spectrometry of the reaction mixtures, spectrophotometric assays and molecular docking. The N-carboxyacyl derivatives were good substrates of pea ALDH10 but were only weakly oxidized by the two plant ALDH7s. The N-phenylalanyl and N-tyrosyl derivatives of 3-aminopropionaldehyde were good substrates of pea and maize ALDH7. Particularly the former compound was converted very efficiently (based on the kcat/Km ratio), but it was only weakly oxidized by pea ALDH10. Although no compound exhibited the same level of substrate properties for both ALDH families, we show that these enzymes may possess more common substrates than expected.

Sterol Extraction from Isolated Plant Plasma Membrane Vesicles Affects H+-ATPase Activity and H+-Transport.

Plasma membrane H+-ATPase is known to be detected in detergent-resistant sterol-enriched fractions, also called "raft" domains. Studies on H+-ATPase reconstituted in artificial or native membrane vesicles have shown both sterol-mediated stimulations and inhibitions of its activity. Here, using sealed isolated plasma membrane vesicles, we investigated the effects of sterol depletion in the presence of methyl-ß-cyclodextrin (MßCD) on H+-ATPase activity. The rate of ATP-dependent ∆µH+ generation and the kinetic parameters of ATP hydrolysis were evaluated. We show that the relative sterols content in membrane vesicles decreased gradually after treatment with MßCD and reached approximately 40% of their initial level in 30 mM probe solution. However, changes in the hydrolytic and H+-transport activities of the enzyme were nonlinear. The extraction of up to 20% of the initial sterols was accompanied by strong stimulation of ATP-dependent H+-transport in comparison with the hydrolytic activity of enzymes. Further sterol depletion led to a significant inhibition of active proton transport with an increase in passive H+-leakage. The solubilization of control and sterol-depleted vesicles in the presence of dodecyl maltoside negated the differences in the kinetics parameters of ATP hydrolysis, and all samples demonstrated maximal hydrolytic activities. The mechanisms behind the sensitivity of ATP-dependent H+-transport to sterols in the lipid environment of plasma membrane H+-ATPase are discussed.

Dynamics of Fusarium Mycotoxins and Lytic Enzymes during Pea Plants' Infection.

Fusarium species are common plant pathogens that cause several important diseases. They produce a wide range of secondary metabolites, among which mycotoxins and extracellular cell wall-degrading enzymes (CWDEs) contribute to weakening and invading the host plant successfully. Two species of Fusarium isolated from peas were monitored for their expression profile of three cell wall-degrading enzyme coding genes upon culturing with extracts from resistant (Sokolik) and susceptible (Santana) pea cultivars. The extracts from Santana induced a sudden increase in the gene expression, whereas Sokolik elicited a reduced expression. The coherent observation was that the biochemical profile of the host plant plays a major role in regulating the fungal gene expression. In order to uncover the fungal characteristics in planta, both pea cultivars were infected with two strains each of F. proliferatum and F. oxysporum on the 30th day of growth. The enzyme activity assays from both roots and rhizosphere indicated that more enzymes were used for degrading the cell wall of the resistant host compared to the susceptible host. The most commonly produced enzymes were cellulase, ß-glucosidase, xylanase, pectinase and lipase, where the pathogen selectively degraded the components of both the primary and secondary cell walls. The levels of beauvericin accumulated in the infected roots of both cultivars were also monitored. There was a difference between the levels of beauvericin accumulated in both the cultivars, where the susceptible cultivar had more beauvericin than the resistant one, showing that the plants susceptible to the pathogen were also susceptible to the toxin accumulation.

A new catalytic mechanism of bacterial ferredoxin-NADP+ reductases due to a particular NADP+ binding mode.

Ferredoxin-NADP+ reductases (FNRs) are ubiquitous flavoenzymes involved in redox metabolisms. FNRs catalyze the reversible electron transfer between NADP(H) and ferredoxin or flavodoxin. They are classified as plant- and mitochondrial-type FNR. Plant-type FNRs are divided into plastidic and bacterial classes. The plastidic FNRs show turnover numbers between 20 and 100 times higher than bacterial enzymes and these differences have been related to their physiological functions. We demonstrated that purified Escherichia coli FPR (EcFPR) contains tightly bound NADP+ , which does not occur in plastidic type FNRs. The three-dimensional structure of EcFPR evidenced that NADP+ interacts with three arginines (R144, R174, and R184) which could generate a very high affinity and structured site. These arginines are conserved in other bacterial FNRs but not in the plastidic enzymes. We have cross-substituted EcFPR arginines with residues present in analogous positions in the Pisum sativum FNR (PsFNR) and replaced these amino acids by arginines in PsFNR. We analyzed all proteins by structural, kinetic, and stability studies. We found that EcFPR mutants do not contain bound NADP+ and showed increased Km for this nucleotide. The EcFPR activity was inhibited by NADP+ but this behavior disappeared as arginines were removed. A NADP+ analog of the nicotinamide portion produced an activating effect on EcFPR and promoted the NADP+ release. Our results give evidence for a new model of NADP+ binding and catalysis in bacterial FNRs.We propose that this tight NADP+ binding constitutes an essential catalytic and regulatory mechanism of bacterial FNRs involved in redox homeostasis.

Effects of Superoxide Dismutase Inhibitors and Glucose on Cell Death and Generation of Reactive Oxygen Species in Pea Leaves.

The effects of superoxide dismutase (SOD) inhibitors, diethyldithiocarbamate (DDC), triethylenetetramine (trien), and their combination with glucose on cells of the epidermis from pea leaves of different age (rapidly growing young leaves and slowly growing old leaves) was investigated. DDC and trien caused death of the guard cells as determined by destruction of their nuclei. Glucose did not affect destruction of the nuclei induced by SOD inhibitors in the cells from old leaves, but intensified it in the cells from young leaves. 2-Deoxyglucose, an inhibitor of glycolysis, and propyl gallate, SOD-mimic and antioxidant, suppressed destruction of the nuclei that was caused by SOD inhibitors and glucose in cells of the epidermis from the young, but not from the old leaves. Glucose and trien stimulated, and propyl gallate reduced generation of reactive oxygen species (ROS) in the pea epidermis as determined by the fluorescence of 2',7'-dichlorofluorescein (DCF). Carbonyl cyanide m-chlorophenylhydrazone (CCCP), a protonophoric uncoupler of oxidative and photosynthetic phosphorylation, suppressed the DCF fluorescence in the guard cells. Treatment of the cells with CCCP followed by its removal with washing increased destruction of the nuclei caused by SOD inhibitors and glucose. In young leaves, CCCP was less effective than in old ones. The findings demonstrate the effects of SOD inhibitors and glucose on the cell death and generation of ROS and could indicate glycolysis-dependent ROS production.

Involvement of plasma membrane H+-ATPase in diamide-induced extracellular alkalization by roots from pea seedlings.

MAIN CONCLUSION: The plasma membrane H+-ATPase can be considered as a redox-dependent enzyme, because diamide-mediated inhibition of its hydrolytic and transport activities is accompanied by alkalization of the rhizosphere and retardation of root growth. Plasma membranes were isolated from roots of etiolated pea seedlings treated in the presence of an oxidant-diamide and an inhibitor of redox-sensitive protein phosphatase-phenylarsine oxide. Hydrolytic and proton transport activities of H+-ATPase were determined. The effects of diamide appeared in inhibition of both ATP hydrolysis and the proton transport. However, root treatment with phenylarsine oxide only slightly reduced Vmax, but did not affect ATP-dependent proton transport. The thiol groups of cysteines in the proteins can act as molecular targets for both compounds. However, treatment of isolated membranes with diamide or dithiothreitol did not have any effect on the H+ transport. It can be assumed that water-soluble diamide acts indirectly and its effects are not associated with oxidation of H+-ATPase cysteines. Therefore, plasmalemma was subjected to PEGylation-process where reduced cysteines available for PEG maleimide (5 kDa) were alkylated. Detection of such cysteines was carried out by Western blot analysis with anti-ATPase antibodies. It was found that shifts in the apparent molecular weight were detected only for denaturated proteins. These data suggest that available thiols are not localized on the enzyme surfaces. BN-PAGE analysis showed that the molecular weights of the ATPase complexes are almost identical in all samples. Therefore, oligomerization is probably not the reason for the inhibition of ATPase activity. Roots treated with these inhibitors in vivo exhibited stunted growth; however, a strong alkaline zone around the roots was formed only in the presence of diamide. Involvement of H+-ATPase redox regulation in this process is discussed.

Cutin:cutin-acid endo-transacylase (CCT), a cuticle-remodelling enzyme activity in the plant epidermis.

Cutin is a polyester matrix mainly composed of hydroxy-fatty acids that occurs in the cuticles of shoots and root-caps. The cuticle, of which cutin is a major component, protects the plant from biotic and abiotic stresses, and cutin has been postulated to constrain organ expansion. We propose that, to allow cutin restructuring, ester bonds in this net-like polymer can be transiently cleaved and then re-formed (transacylation). Here, using pea epicotyl epidermis as the main model, we first detected a cutin:cutin-fatty acid endo-transacylase (CCT) activity. In-situ assays used endogenous cutin as the donor substrate for endogenous enzymes; the exogenous acceptor substrate was a radiolabelled monomeric cutin-acid, 16-hydroxy-[3H]hexadecanoic acid (HHA). High-molecular-weight cutin became ester-bonded to intact [3H]HHA molecules, which thereby became unextractable except by ester-hydrolysing alkalis. In-situ CCT activity correlated with growth rate in Hylotelephium leaves and tomato fruits, suggesting a role in loosening the outer epidermal wall during organ growth. The only well-defined cutin transacylase in the apoplast, CUS1 (a tomato cutin synthase), when produced in transgenic tobacco, lacked CCT activity. This finding provides a reference for future CCT protein identification, which can adopt our sensitive enzyme assay to screen other CUS1-related enzymes.

High-Light versus Low-Light: Effects on Paired Photosystem II Supercomplex Structural Rearrangement in Pea Plants.

In plant grana thylakoid membranes Photosystem II (PSII) associates with a variable number of antenna proteins (LHCII) to form different types of supercomplexes (PSII-LHCII), whose organization is dynamically adjusted in response to light cues, with the C2S2 more abundant in high-light and the C2S2M2 in low-light. Paired PSII-LHCII supercomplexes interacting at their stromal surface from adjacent thylakoid membranes were previously suggested to mediate grana stacking. Here, we present the cryo-electron microscopy maps of paired C2S2 and C2S2M2 supercomplexes isolated from pea plants grown in high-light and low-light, respectively. These maps show a different rotational offset between the two supercomplexes in the pair, responsible for modifying their reciprocal interaction and energetic connectivity. This evidence reveals a different way by which paired PSII-LHCII supercomplexes can mediate grana stacking at diverse irradiances. Electrostatic stromal interactions between LHCII trimers almost completely overlapping in the paired C2S2 can be the main determinant by which PSII-LHCII supercomplexes mediate grana stacking in plants grown in high-light, whereas the mutual interaction of stromal N-terminal loops of two facing Lhcb4 subunits in the paired C2S2M2 can fulfil this task in plants grown in low-light. The high-light induced accumulation of the Lhcb4.3 protein in PSII-LHCII supercomplexes has been previously reported. Our cryo-electron microscopy map at 3.8 Å resolution of the C2S2 supercomplex isolated from plants grown in high-light suggests the presence of the Lhcb4.3 protein revealing peculiar structural features of this high-light-specific antenna important for photoprotection.

Salicylate-Induced Chitinases in Pea Roots.

Three proteins induced by salicylic acid were revealed in pea roots. These proteins were identified as chitinase isozymes belonging to the glycoside hydrolases family 18. The PsCam050724 transcript encoding at least one of these isoforms was found, allowing us to determine its primary structure, which lacks the signal peptide.

Key volatile off-flavor compounds in peas (Pisum sativum L.) and their relations with the endogenous precursors and enzymes using soybean (Glycine max) as a reference.

The dominant volatile off-flavor compounds of pea and soy milk were investigated by gas chromatography-olfactometry-mass spectrometry (GC-O-MS), sensory evaluation, and odor-activity values (OAVs), which led to the identification of their differences. We identified 11 aroma compounds as important odorants with OAVs greater than 1 in pea and soy milk. OAVs contribution rate demonstrated that 6 compounds contributed most to the characteristic off-flavor of pea milk, among which 2-methoxy-3-isopropyl-(5 or 6)-methyl pyrazine, hexanal, (E,E)-2,4-nonadienal, and (E,E)-2,4-decadienal contributed more than others. For soy milk, 1-octen-3-one, hexanal, (E,E)-2,4-nonadienal, and (E,E)-2,4-decadienal showed more important contributions. These odor-active compounds were divided into non-lipoxygenase (non-LOX) and LOX pathways based on their synthesis. Several endogenous enzymes that are important to the LOX pathway were identified by liquid chromatography tandem mass spectrometry (LC-MS/MS), and the contents of key off-flavor compounds were found to be related to the enzyme activities, while the lipid content was not an important factor.

Cytochrome oxidase and alternative oxidase pathways of mitochondrial electron transport chain are important for the photosynthetic performance of pea plants under salinity stress conditions.

The flexible plant mitochondrial electron transport chain with cytochrome c oxidase (COX) and alternative oxidase (AOX) pathways is known to be modulated by abiotic stress conditions. The effect of salinity stress on the mitochondrial electron transport chain and the importance of COX and AOX pathways for optimization of photosynthesis under salinity stress conditions is not clearly understood. In the current study, importance of COX and AOX pathways for photosynthetic performance of pea plants (Pisum sativum L. Pea Arkel cv) was analysed by using the mitochondrial electron transport chain inhibitors Antimycin A (AA) and salicylhydroxamic acid (SHAM) which restrict the electron flow through COX and AOX pathways respectively. Salinity stress resulted in decreased CO2 assimilation rates, leaf stomatal conductance, transpiration and leaf intercellular CO2 concentration in a stress dependent manner. Superimposition of leaves of salt stressed plants with AA and SHAM caused cellular H2O2 and O2- accumulation along with cell death. Additionally, aggravation in decrease of CO2 assimilation rates, leaf stomatal conductance, transpiration and leaf intercellular CO2 concentration upon superimposition with AA and SHAM during salinity stress suggests the importance of mitochondrial oxidative electron transport for photosynthesis. Increased expression of AOX1a and AOX2 transcripts along with AOX protein levels indicated up regulation of AOX pathway in leaves during salinity stress. Chlorophyll fluorescence measurements revealed enhanced damage to Photosystem (PS) II in the presence of AA and SHAM during salinity stress. Results suggested the beneficial role of COX and AOX pathways for optimal photosynthetic performance in pea leaves during salinity stress conditions.

An innovative artificial photosystem II constructed from PSII core of Thermosynechococcus vulcanus and LHCII of Pisum sativum - A new approach for studying the function of photosynthetic antenna.

In photosynthesis, the antenna system captures solar energy and transfers the excitations to photosystem II (PSII) core complex where charge separation, water splitting and oxygen evolution occur. In the evolution of photosynthesis from aquatic to terrestrial environments, the structure of PSII core complex was highly conserved while a variety of antenna forms became differentiated. In order to study the principles for energy transport from antenna to the PSII reaction center, we have explored whether the major light harvesting complex of PSII (LHCII) of higher plants can transfer energy to the cyanobacteria PSII core complexes (CC). For this purpose, LHCII from pea and CC from Thermosynechococcus vulcanus were isolated and co-reconstituted into liposome at LHCII:CC molar ratios of 2:1, 4:1 and 6:1, respectively. Chemical-cross linking followed by LC-MS/MS analysis confirmed the biochemical interaction between LHCII and CC in the liposome membrane. The analyses of 77 K fluorescence emission spectra and antenna cross section of PSII indicated that LHCII can transfer energy directly to the cyanobacterial CC. The study has laid the basis for further research on the mechanism of energy transfer from LHCII to PSII CC. This result may also open a new possibility for design and development of new artificial PSII in the application of solar energy conversion.

A mild intensity of the enzyme-support multi-point attachment promotes the optimal stabilization of mesophilic multimeric enzymes: Amine oxidase from Pisum sativum.

Stabilization of dimeric enzymes requires the stabilization of the quaternary structure as well as the 3D one. Both subunits may be easily immobilized on a highly activated support. Additional stabilization of the 3D structure may be achieved via multipoint covalent attachment (MCA) on highly activated supports. In the case of monomeric enzymes or thermophilic dimeric ones, the optimal stabilization is obtained via the most intense MCA and it is associated to a small loss of catalytic activity. However, in the case of mesophilic enzymes, a very intense MCA of both subunits may promote negative effects, e.g., associated to distortions of the assembly between subunits and a subsequent very important loss of catalytic activity. A dimeric mesophilic amine oxidase from P.sativum was stabilized by MCA on glyoxyl-agarose. Both subunits were covalently immobilized on the support through the region with the highest density in Lys residues. In addition to that, an interesting activity/stabilization binomial was obtained after only 3 h of enzyme-support multiinteraction (50 % of activity/350 fold stabilization). However, after 24 h of enzyme-support multi-interaction this binomial activity-stabilization decreased down to 30/150. A moderate multiinteraction seems to be the optimal strategy for immobilization-stabilization of mesophilic dimeric enzymes and it promotes moderate losses of activity and interesting stabilizations against the combined effect of heat, acid pH and ethanol. The control of the intensity of enzyme-support multi-interactions becomes now strictly necessary.

Novel approach using activated cellulose film for efficient immobilization of purified diamine oxidase to enhance enzyme performance and stability.

The presence of various contaminants in foodstuffs has led to serious public health concerns. Diamine oxidase (DAO) has attracted tremendous attention for guarding food safety as well as clinical and environmental industries. In this study, DAO from Pisum sativum (Pea) seedlings was extracted and purified by dialysis and gel filtration. Purified DAO was covalently immobilized onto the surface of nitrocellulose membrane using glutaraldehyde. The obtained bioaffinity support has efficiently shown high yield immobilization of DAO from pea seedlings. The optimal conditions of free and immobilized DAO activity were evaluated against the substrate, Putrescine dihydrochloride. The influence of pH, temperature, storage stability, and reusability of immobilized enzyme with comparison to the free enzyme was studied and the results showed that the stabilities were significantly enhanced compared with free counterpart. Residual activity of the immobilized enzyme was 59% of the initial activity after being recycled 10 times. We approve that this novel low cost immobilized DAO carrier presents a new approach in large scale applications.

Stoichiometry of two plant glycine decarboxylase complexes and comparison with a cyanobacterial glycine cleavage system.

The multienzyme glycine cleavage system (GCS) converts glycine and tetrahydrofolate to the one-carbon compound 5,10-methylenetetrahydrofolate, which is of vital importance for most if not all organisms. Photorespiring plant mitochondria contain very high levels of GCS proteins organised as a fragile glycine decarboxylase complex (GDC). The aim of this study is to provide mass spectrometry-based stoichiometric data for the plant leaf GDC and examine whether complex formation could be a general property of the GCS in photosynthesizing organisms. The molar ratios of the leaf GDC component proteins are 1L2 -4P2 -8T-26H and 1L2 -4P2 -8T-20H for pea and Arabidopsis, respectively, as determined by mass spectrometry. The minimum mass of the plant leaf GDC ranges from 1550 to 1650 kDa, which is larger than previously assumed. The Arabidopsis GDC contains four times more of the isoforms GCS-P1 and GCS-L1 in comparison with GCS-P2 and GCS-L2, respectively, whereas the H-isoproteins GCS-H1 and GCS-H3 are fully redundant as indicated by their about equal amounts. Isoform GCS-H2 is not present in leaf mitochondria. In the cyanobacterium Synechocystis sp. PCC 6803, GCS proteins concentrations are low but above the complex formation threshold reported for pea leaf GDC. Indeed, formation of a cyanobacterial GDC from the individual recombinant GCS proteins in vitro could be demonstrated. Presence and metabolic significance of a Synechocystis GDC in vivo remain to be examined but could involve multimers of the GCS H-protein that dynamically crosslink the three GCS enzyme proteins, facilitating glycine metabolism by the formation of multienzyme metabolic complexes. Data are available via ProteomeXchange with identifier PXD018211.

In vitro determination of diamine oxidase activity in food matrices by an enzymatic assay coupled to UHPLC-FL.

Intestinal diamine oxidase (DAO) acts as a protective barrier against exogenous histamine. A deficit of DAO activity can lead to the appearance of histamine intolerance, a clinical condition that may be treated by a low-histamine diet and oral DAO supplementation to enhance intestinal histamine degradation. As sources of DAO, porcine kidneys and certain legume seedlings are suitable components for the formulation of a DAO supplement. The aim of this work was to develop a rapid and reliable methodology for the in vitro determination of DAO activity in food matrices based on an enzymatic assay coupled to UHPLC-FL. The proposed method showed a satisfactory linearity and sensitivity and provided a relative standard deviation lower than 3%, guaranteeing method precision, and a mean recovery greater than 99% both for lyophilized pea sprouts and porcine kidney protein extracts. A high specificity is a key attribute of this method due to the use of histamine as the reaction substrate and the direct quantification of its degradation. Moreover, the lack of interference of catalase and hydrogen peroxide is another advantage in comparison with previously published methods. Lyophilized pea sprouts showed the greatest histamine-degrading activity (0.40 ± 0.01 mU/mg), followed by porcine kidney protein extracts (0.23 ± 0.01 mU/mg) and commercial DAO supplements (0.09 ± 0.06 mU/mg). This technique could be used as a tool to validate the DAO activity of food matrices of potential interest for the treatment of histamine intolerance.

Purification and Partial Characterization of Peroxidases from Three Food Waste By-Products: Broad Bean Pods, Pea Pods, and Artichoke Stems.

In this study, peroxidases (PODs) from three waste by-products: broad bean pods (BBP), pea pods (PP), and artichoke stems (ARS) were purified and their optimal conditions were determined for the first time. The purification process resulted in 4.32, 7.21, and 8.9% of POD recoveries for PP, ARS, and BBP, respectively. They were purified 2.12-, 32.97-, and 10-fold with specific activities of 27.26, 266.43, and 27 U/mg of protein, respectively. Analysis of their optimal conditions showed that POD purified from BBP and PP exhibited the highest activity at 60 °C temperature and pH 6 and 8 with strong affinity with catechol substrate (Km of 0.356 and 0.189 mM; Vmax of 0.08 and 0.041 µM/min for BBP and PP, respectively). The highest activity of ARS POD was obtained under the following conditions: temperature at 50 °C, pH from 6 to 8, and guaiacol as substrate (Km 0.375 mM; Vmax 0.012 µM/min). Apart from giving the opportunity for recycling the food industry wastes, the studied waste by-products could represent an alternative source of PODs that could find several applications in the biotechnological, chemical, and food industries.

Oxidation of imidazole- and pyrazole-derived aldehydes by plant aldehyde dehydrogenases from the family 2 and 10.

Plant cytosolic aldehyde dehydrogenases from family 2 (ALDH2s, EC 1.2.1.3) are non-specific enzymes and participate for example in the metabolism of acetaldehyde or biosynthesis of phenylpropanoids. Plant aminoaldehyde dehydrogenases (AMADHs, ALDH10 family, EC 1.2.1.19) are broadly specific and play an important role in polyamine degradation or production of osmoprotectants. We have tested imidazole and pyrazole carbaldehydes and their alkyl-, allyl-, benzyl-, phenyl-, pyrimidinyl- or thienyl-derivatives as possible substrates of plant ALDH2 and ALDH10 enzymes. Imidazole represents a building block of histidine, histamine as well as certain alkaloids. It also appears in synthetic pharmaceuticals such as imidazole antifungals. Biological compounds containing pyrazole are rare (e.g. pyrazole-1-alanine and pyrazofurin antibiotics) but the ring is often found as a constituent of many synthetic drugs and pesticides. The aim was to evaluate whether aldehyde compounds based on azole heterocycles are oxidized by the enzymes, which would further support their expected role as detoxifying aldehyde scavengers. The analyzed imidazole and pyrazole carbaldehydes were only slowly converted by ALDH10s but well oxidized by cytosolic maize ALDH2 isoforms (particularly by ALDH2C1). In the latter case, the respective Km values were in the range of 10-2000 µmol l-1; the kcat values appeared mostly between 0.1 and 1.0 s-1. The carbaldehyde group at the position 4 of imidazole was oxidized faster than that at the position 2. Such a difference was not observed for pyrazole carbaldehydes. Aldehydes with an aromatic substituent on their heterocyclic ring were oxidized faster than those with an aliphatic substituent. The most efficient of the tested substrates were comparable to benzaldehyde and p-anisaldehyde known as the best aromatic aldehyde substrates of plant cytosolic ALDH2s in vitro.

Regulation of two GTPases Toc159 and Toc34 in the translocon of the outer envelope of chloroplasts.

The GTPases Toc159 and Toc34 of the translocon of the outer envelope of chloroplasts (TOC) are involved in recognition and transfer of precursor proteins at the cytosolic face of the organelle. Both proteins engage multiple interactions within the translocon during the translocation process, including dimeric states of their G-domains. The units of the Toc34 homodimer are involved in the recognition of the transit peptide representing the translocation signal of precursor proteins. This substrate recognition is part of the regulation of the GTPase cycle of Toc34. The Toc159 monomer and the Toc34 homodimer recognize the transit peptide of the small subunit of Rubisco at the N- and at the C-terminal region, respectively. Analysis of the transit peptide interaction by crosslinking shows that the heterodimer between both G-domains binds pSSU most efficiently. While substrate recognition by Toc34 homodimer was shown to regulate nucleotide exchange, we provide evidence that the high activation energy of the GTPase Toc159 is lowered by substrate recognition. The nucleotide affinity of Toc34G homodimer and Toc159G monomer are distinct, Toc34G homodimer recognizes GDP and Toc159G GTP with highest affinity. Moreover, the analysis of the nucleotide association rates of the monomeric and dimeric receptor units suggests that the heterodimer has an arrangement distinct from the homodimer of Toc34. Based on the biochemical parameters determined we propose a model for the order of events at the cytosolic side of TOC. The molecular processes described by this hypothesis range from transit peptide recognition to perception of the substrate by the translocation channel.

Purification of a non-specific nucleoside hydrolase from Alaska pea seeds.

A non-specific nucleoside hydrolase has been isolated from germinated Alaska pea seeds. The enzyme catalyzes the hydrolysis of both purines and pyrimidines along with ribo- and deoxyribonucleosides. A purification scheme utilized ammonium sulfate precipitation, ion exchange chromatography and size exclusion chromatography, resulted in 103-fold purification with a recovery of 2.8%. The purified protein has a specific activity of 0.308 µmol/min•mg. The subunit molecular weight was 26103 Da and the enzyme exists as a dimer. The enzyme retains a significant amount of activity over a wide pH range with the maximum activity occurring at a pH of 6.0. The maximum activity was observed with adenosine as the substrate followed by inosine and guanosine, respectively. The Km for adenosine was 184 ±â€¯34 µM and for inosine 283 ±â€¯88 µM. In addition to the nucleoside hydrolase activity, adenosine deaminase activity was seen in the initial extract. Using adenosine as the substrate with the initial extract from the germinated seeds, the products adenine, inosine, and hypoxanthine were identified based on their retention times during reverse phase HPLC.