Structure of soybean β-cyanoalanine synthase and the molecular basis for cyanide detoxification in plants

Plants produce cyanide (CN-) during ethylene biosynthesis in the mitochondria and require β-cyanoalanine synthase (CAS) for CN- detoxification. Recent studies show that CAS is a member of the β-substituted alanine synthase (BSAS) family, which also includes the Cys biosynthesis enzyme O-acetylserine sulfhydrylase (OASS), but how the BSAS evolved distinct metabolic functions is not understood. Here we show that soybean (Glycine max) CAS and OASS form α-aminoacrylate reaction intermediates from Cys and O-acetylserine, respectively. To understand the molecular evolution of CAS and OASS in the BSAS enzyme family, the crystal structures of Gm-CAS and the Gm-CAS K95A mutant with a linked pyridoxal phosphate (PLP)-Cys molecule in the active site were determined. These structures establish a common fold for the plant BSAS family and reveal a substrate-induced conformational change that encloses the active site for catalysis. Comparison of CAS and OASS identified residues that covary in the PLP binding site. The Gm-OASS T81M, S181M, and T185S mutants altered the ratio of OASS:CAS activity but did not convert substrate preference to that of a CAS. Generation of a triple mutant Gm-OASS successfully switched reaction chemistry to that of a CAS. This study provides new molecular insight into the evolution of diverse enzyme functions across the BSAS family in plants.     

β-Glucosidase activity is involved in scent production in Narcissus flowers

Extrinsic environmental cues and intrinsic developmental stages of the flower control the production of scent from flowers. Flowers emit scent only when they are open; yet, the precursors for the aromatic compounds are also present in buds, stored as non-fragrant glycosides in the vacuole. We demonstrate that in Narcissus flowers scent emission is concurrent with an increase in the activity of β-glucosidase. The inhibition in vivo of β-glucosidase activity decreases scent emission from Narcissus flowers. The β-glucosidase activity was partially purified and the K_m, V_max and inhibition by gluconic acid lactone was determined.     

Regulation of β-exoglucanase activity production by Saccharomyces cerevisiae in batch and continuous culture

The rate of synthesis and secretion of exo-1–3--glucanase activity closely paralleled the specific rate of growth in exponentially growing Saccharomyces cerevisiae cells in batch culture. When the stationary phase was reached both synthesis and secretion stopped. No activity was synthesized when the cells were maintained in carbon sources that did not allow them to grow. Studies in continuous culture indicate a strong relationship between the synthesis of exoglucanase activity and the specific growth rate. These results are taken as evidence of an essential role of this activity during the yeast budding cycle.Non-standard abbreviations p-NPG p-nitrophenyl--d-glucopyranoside - S_r glucose concentration in the sterile reservoir; , glucose concentration at the steady state - biomass density at the steady state - G glucanase activity - Q _g specific exoglucanase synthesis rate     

Factors influencing β-glucosidase production, activity and stability inNectria catalinensis

The influence of different cultivation conditions on β-glucosidase production and of some parameters on the activity and stability of this enzyme were studied inNectria catalinensis. Maximal β-glucosidase production was achieved with ammonium nitrate (0.5 g N/L) as nitrogen source. Tween 80, Tween 20 and Triton X-100 increased β-glucosidase yields, Tween 80 was the most effective for enzyme release and growth at a concentration of 3.4 mmol/L. On the other hand, Tween 20 and Triton X-100 had an inhibitory effect onN. catalinensis growth. A temperature of 23°C and an initial pH of cultures of 6.5 were optimal for biomass and β-glucosidase production. Under optimal cultural conditions (ammonium nitrate, 0.5 g N/L; Tween 80, 3.4 mmol/L; 23°C; initial pH 6.5) the β-glucosidase yield was increased more than five fold respect to the initial state. Optimal temperature for β-glucosidase activity was 45°C, the initial activity dropped 60 % after 6 h of incubation at this temperature. Optimal pH for enzyme activity was 5.3. At this pH the β-glucosidase was completely stable after 3 d of incubation. TheV andK _m values calculated from Lineweaver-Burk and Eadie-Hofstee plots were 0.23 μmol 4-nitrophenol per min per mg of protein and 0.25 mmol 4-nitrophenol β-d-glucopyranoside per L, respectively. The activation energy according to Arrhenius plot was 49.6 KJ/mol.     

Stereochemistry of the β-cyanoalanine synthetase and S-alkylcysteine lyase reactions

The stereochemistry of the replacement of the SH-group of cysteine by CN catalyzed by β-cyanoalanine synthetase was studied using cysteine stereospecifically tritiated at C-3. Analysis of the resulting β-cyanoalanine by conversion into fumarate via aspartate and malate showed that the reaction had occurred with retention of configuration at C-3. Using cystine stereospecifically labeled at C-3 with tritium or with tritium and deuterium, it was found that the α,β-elimination reaction catalyzed by S-alkylcysteine lyase involves stereo-specific replacement of the β-substituent of the substrate by a hydrogen derived from the solvent, D₂O or H₂O, with retention of configuration to give pyruvate containing a chiral methyl group. The results are discussed, particularly in the light of mechanistic proposals by Braunstem and co-workers.     

The Role of β-Hydroxypropionate in Ethylene Biosynthesis

To search precursors of ethylene in banana fruits, ethylene formation from acetate-2-¹⁴C and fumarate-2,3-¹⁴C by banana slices was studied. Ethylene-¹⁴C formation from acetate-2-^l4C was reduced by the addition of malonate or β-hydroxypropionate, and it was enhanced in a sealed chamber in comparison with the case in an aeration chamber. No label of fumarate-2,3-¹⁴C was incorporated into ethylene.

From these facts it was suggested that acetate-2-¹⁴C was incorporated into ethylene via malonate and β-hydroxypropionate. Participation of fumarate in ethylene biosynthesis of banana fruits was ruled out. β-Hydroxypropionate was postulated as an effective precursor of ethylene formation from acetate-2-^l4C.     

Formation of β-cyanoalanine and pyruvate by Acacia georginae

Pyruvate is formed on incubation of l-cysteine with acetone powder preparations of Acacia georginae but in the presence of cyanide, β-cyanoalanine is produced and pyruvate production is highly depressed. The pH optimum for pyruvate production is 8·5. In the presence of fluoride (1·5 mM), the pH profile is unchanged and in the presence of cyanide (1·5 mM), minimal pyruvate production occurs at pH 8·5. Although addition of pyridoxal phosphate had no influence on pyruvate or β-Cyanoalanine production, these processes were prevented by sodium borohydride, an inhibitor of pyridoxal enzymes. Neither l-serine nor O-acetyl-l-serine serve as alternative substrates for pyruvate production. β-Fluoroalanine was not detected on incubating fluoride with an enzyme preparation from A. georginae acetone powders.     

Mapping of β-glucan content and β-glucanase activity loci in barley grain and malt

Genetic study of -glucan content and -glucanase activity has been facilitated by recent developments in quantitative trait loci (QTL) analysis. QTL for barley and malt -glucan content and for green and finished malt -glucanase activity were mapped using a 123-point molecular marker linkage map from the cross of Steptoe/Morex. Three QTL for barley -glucan, 6 QTL for malt -glucan, 3 QTL for -glucanase in green malt and 5 QTL for -glucanase in finished malt were detected by interval mapping procedures. The QTL with the largest effects on barley -glucan, malt glucan, green malt -glucanase and finished malt glucanase were identified on chromosomes 2,1,4 and 7, respectively. A genome map-based approach allows for dissection of relationships among barley and malt glucan content, green and finished malt -glucanase activity, and other malting quality parameters.     

Carboxydismutase activity in plants with and without β-carboxylation photosynthesis

Summary A comparison was made of the activity of carboxydismutase (ribulose-1,5-diphosphate carboxylase) between higher plant species which possess the -carboxylation (C₄-dicarboxylic acid) pathway for photosynthesis and species which lack this pathway. Contrary to earlier findings no marked difference in the level of this enzyme was found between the two groups of species. Chloroplast-containing vascular-bundle-sheath cells which seem to be present only in plants with -carboxylation apparently contain relatively high carboxydismutase activity.C.I.W.-D.P.B. Publ. No. 453.     

Metal resistance in Acinetobacter and its relation to β-lactamase production

Thirty nine clinical isolates of Acinetobacter belonging to six species were tested for resistance to 20 metal ions and their ability to produce -lactamase. Fifty two percent of the strains produced -lactamase. -Lactamase producers and non-producers were almost equally distributed in the different species. A. baumannii was the predominant biotype and was found to be most resistant to metals. Resistance to mercury was prevalent in -lactamase-producing A. baumannii only. Silver resistant strains of A. baumannii produced -lactamase. Sensitivity and resistance to copper and cadium was equally distributed between -lactamase producers and non-producers. -Lactamase-producer and -non-producer strains were uniformly sensitive to cadmium except Acinetobacter genospecies 1.     

δ-endotoxin activity and spore production in batch and fed-batch cultures of Bacillus thuringiensis

Summary The toxicity and the spore count of batch and fed batch cultures of Bacillus thuringiensis var. israelensis were studied. Spore counts reached in both batch and fed batch cultures were as high as those reported in the literature, but the levels of toxicity found in the latter were about one order of magnitude lower than those attained in batch cultures. Avoiding restricted cultures might be necessary to reach high titres of -endotoxin, which are essential if a good product is intended. Furthermore, spore count might not be a good parameter to predict insecticidal activity of Bacillus thuringiensis cultures.     

In vitro production of flowering shoots in ‘German Red’ carnation: effect of uniconazole and gibberellic acid

Summary Callus regenerated near the base of senescing petals of flower bud explants of German Red carnation (Dianthus caryophyllus L.) produced adventitious flowering microshoots on MS-medium containing benzylaminopurine (8.9 M) and naphthaleneacetic acid (2.7 M). When these microshoots were subcultured with some callus, additional adventitious flowering microshoots were produced from the callus. The production of adventitious flowering shoots continued for many subcultures spanning a period of more than two years. Uniconazole (6.9 M) increased the number of adventitious shoots formed by as much as two-fold but decreased shoot length by about 50%. In contrast, GA₃ (2.9 M) decreased adventitious shoot formation and increased shoot length. Regardless of the growth regulator treatment, virtually all of the adventitious shoots produced flower buds. Thus, the growth regulators influenced flowering only indirectly by altering the number of adventitious shoots produced in vitro. These results demonstrate that the flowering habit of the adventitious shoots of German Red carnation is highly persistent and the flowering stimulus continues to be transmitted to the newly formed microshoots through the callus.     

PKR downregulation prevents neurodegeneration and β-amyloid production in a thiamine-deficient model

Brain thiamine homeostasis has an important role in energy metabolism and displays reduced activity in Alzheimer''s disease (AD). Thiamine deficiency (TD) induces regionally specific neuronal death in the animal and human brains associated with a mild chronic impairment of oxidative metabolism. These features make the TD model amenable to investigate the cellular mechanisms of neurodegeneration. Once activated by various cellular stresses, including oxidative stress, PKR acts as a pro-apoptotic kinase and negatively controls the protein translation leading to an increase of BACE1 translation. In this study, we used a mouse TD model to assess the involvement of PKR in neuronal death and the molecular mechanisms of AD. Our results showed that the TD model activates the PKR-eIF2α pathway, increases the BACE1 expression levels of Aβ in specific thalamus nuclei and induces motor deficits and neurodegeneration. These effects are reversed by PKR downregulation (using a specific inhibitor or in PKR knockout mice).Thiamine (vitamin B1) deficiency (TD) induces regionally selective neurodegeneration in the mammal''s brains, particularly in specific thalamus nuclei (submedial thalamus nuclei (SmTN) and ventral lateral nuclei (VLN)) and cerebellum.^{1, 2} TD-induced neuronal loss is associated with a chronic impairment of oxidative metabolism and neuroinflammation associated with glial activation.^{3, 4, 5} Diminished thiamine-dependent processes in humans is not only associated with Wernicke–Korsakoff syndrome but also accompany other neurodegenerative disorders, such as Alzheimer''s disease (AD).⁶ Experimental TD has been used to model the pathogenesis and investigate the cellular mechanisms of neurodegenerative disorders. In mice, TD-induced oxidative stress enhances Aβ accumulation by regulating β-site APP-cleaving enzyme 1 (BACE1) maturation. These effects are amplified in AD mouse model.^{7, 8}The double-stranded RNA-dependent protein kinase (PKR) is one of the four mammalian serine–threonine kinases—the others being HRI (heme-regulated eukaryotic translation initiation factor-2α (eIF2α) kinase), GCN2 (general control nonderepressible 2) and PERK (protein kinase RNA-like endoplasmic reticulum kinase)—that catalyzes the phosphorylation of the α subunit of eIF2 in response to stress signals, leading to an inhibition of protein synthesis.^{9, 10} Activation of PKR is induced by various triggers such as viral infection and endoplasmic reticulum or oxidative stresses^{11, 12} and could be controlled by an interaction with its specific activator PACT (PKR activator), also named RAX in rodents. PKR phosphorylation is known to have a significant role in AD,¹³ and cerebrospinal fluid (CSF) PKR levels could be used as a potential diagnostic biomarker in AD patients.^{14, 15} PKR is a proapoptotic kinase¹⁶ and can exert a number of toxic effects on neurons that could contribute to the functional and pathological alterations in AD brains. PKR also contributes to neurodegeneration and to the pathological molecular mechanisms observed in AD. PKR can mediate Tau phosphorylation induced by Aβ exposure in cell cultures.¹⁷ Additionally, several investigators have demonstrated that eIF2α phosphorylation, via PKR-induced cellular stress, leads to increased BACE1 mRNA translation when, paradoxically, global protein translation is inhibited.^{18, 19, 20, 21} These alterations of BACE1 translational control could be explained by a stress-dependent phenomenon of translation initiation.^{22, 23, 24} Moreover, PKR and eIF2α are highly phosphorylated in SmTN and the cerebellum of TD mouse model. Analyses performed on cerebellar granule neurons exposed to a thiamine metabolic antagonist suggest that TD-induced neuronal death could be partially mediated by PKR activation.²⁵To date, all studies that have explored the deleterious role of PKR activation in neurodegeneration indicate that inhibition of PKR is a promising approach to inhibit pathological mechanisms. Moreover, recent studies have shown that the genetic lack of PKR enhances learning and memory in several behavioral tasks while increasing network excitability.²⁶ The goal of this study was to assess the role of PKR downregulation on neurodegeneration and Aβ production in a mouse model of neurodegeneration.     

Chalcone synthesis and hydroxylation of flavonoids in 3′-position with enzyme preparations from flowers of Dianthus caryophyllus L. (carnation)

Chalcone synthase activity was demonstrated in enzyme preparations from flowers of defined genotypes of Dianthus caryophyllus L. (carnation). In the absence of chalcone isomerase activity, which could be completely excluded by genetic methods, the first product formed from malonyl-CoA and 4-coumaroyl-CoA proved to be naringenin chalcone, followed by formation of naringenin as a result of chemical cyclization. In the presence of chalcone isomerase activity, however, naringenin was the only product of the synthase reaction. In vitro, both 4-coumaryl-CoA and caffeoyl-CoA were found to be used as substrates for the condensation reaction with respective pH optima of 8.0 and 7.0. The results of chemogenetic and enzymatic studies, however, showed that in vivo only 4-coumaroyl-CoA serves as substrate for the formation of the flavonoid skeleton. In confirmation of these results, an NADPH-dependent microsomal 3-hydroxylase activity could be demonstrated, catalyzing hydroxylation of naringenin and dihydrokaempferol in 3-position. Furthermore, a strict correlation was found between 3-hydroxylase activity and the gene r which is known to control the formation of 3, 4-hydroxylated flavonoid compounds.     

A phycocyanin·phellandrene synthase fusion enhances recombinant protein expression and β-phellandrene (monoterpene) hydrocarbons production in Synechocystis (cyanobacteria)

Cyanobacteria can be exploited as photosynthetic platforms for heterologous generation of terpene hydrocarbons with industrial applications. Transformation of Synechocystis and heterologous expression of the β-phellandrene synthase (PHLS) gene alone is necessary and sufficient to confer to Synechocystis the ability to divert intermediate terpenoid metabolites and to generate the monoterpene β-phellandrene during photosynthesis. However, terpene synthases, including the PHLS, have a slow K_cat (low V_max) necessitating high levels of enzyme concentration to enable meaningful rates and yield of product formation. Here, a novel approach was applied to increase the PHLS protein expression alleviating limitations in the rate and yield of β-phellandrene product generation. Different PHLS fusion constructs were generated with the Synechocystis endogenous cpcB sequence, encoding for the abundant in cyanobacteria phycocyanin β-subunit, expressed under the native cpc operon promoter. In one of these constructs, the CpcB·PHLS fusion protein accumulated to levels approaching 20% of the total cellular protein, i.e., substantially higher than expressing the PHLS protein alone under the same endogenous cpc promoter. The CpcB·PHLS fusion protein retained the activity of the PHLS enzyme and catalyzed β-phellandrene synthesis, yielding an average of 3.2 mg product g⁻¹ dry cell weight (dcw) versus the 0.03 mg g⁻¹ dcw measured with low-expressing constructs, i.e., a 100-fold yield improvement. In conclusion, the terpene synthase fusion-protein approach is promising, as, in this case, it substantially increased the amount of the PHLS in cyanobacteria, and commensurately improved rates and yield of β-phellandrene hydrocarbons production in these photosynthetic microorganisms.     

β-1,3-glucanase activity in the stigma of healthy petunia flowers

β-1,3-Glucanase activity was detected in extracts of different tissues of healthy mature petunia flowers except the filament. The stigma was studied further as it had the highest enzyme activity and there is a paucity of information on the occurrence of this enzyme in this tissue. Specific activity of the enzyme was found to increase within the stigmatic tissue from early development until just before anthesis. Following non-denaturing polyacrylamide gel electrophoresis at pH 8.8, extracts of dehiscent stigma seem to contain three acidic isoforms of β-1,3-glucanase. Crude extracts of stigma was passed through a pachyman affinity column. A fraction of affinity-purified active β-1,3-glucanase enzyme was found to have no antifungal activity against Trichoderma viride, Phloma clematidina and Cladosporium fulvum.     

Is a cysteine proteinase inhibitor involved in the regulation of petal wilting in senescing carnation (Dianthus caryophyllus L.) flowers?

Senescence of carnation petals is accompanied by autocatalytic ethylene production and wilting of the petals; the former is caused by the expression of 1-aminocyclopropane-1-carboxylate (ACC) synthase and ACC oxidase genes and the latter is related to the expression of a cysteine proteinase (CPase) gene. CPase is probably responsible for the degradation of proteins, leading to the decomposition of cell components and resultant cell death during the senescence of petals. The carnation plant also has a gene for the CPase inhibitor (DC-CPIn) that is expressed abundantly in petals at the full opening stage of flowers. In the present study, DC-CPIn cDNA was cloned and expressed in E. coli. The recombinant DC-CPIn protein completely inhibited the activities of a proteinase (CPase) extracted from carnation petals and papain. Northern blot analysis showed that the mRNA for CPase (DC-CP1) accumulated in large amounts, whereas that for DC-CPIn disappeared, corresponding to the onset of petal wilting in flowers undergoing natural senescence and exogenous ethylene-induced senescence. Based on these findings, a role of DC-CPIn in the regulation of petal wilting is suggested; DC-CPIn acts as a suppressor of petal wilting, which probably functions to fine-tune petal wilting in contrast to coarse tuning, the up-regulation of CPase activity by gene expression.     

Mycelia-associated β-galactosidase activity in microbial pellets of Aspergillus and Penicillium strains

Pellet formation and production of mycelia-associated -galactosidase were investigated in 15 Aspergillus and Penicillium strains. Mycelia-associated enzyme activity was measured in sonicated homogenates. The properties of the mycelia-associated -galactosidase of A. phoenicis QM 329 was investigated. The pH optimum of the mycelia-associated enzyme was 4.0. The optimum temperature under assay conditions was 70°C and the optimum temperature for repeated lactose hydrolysis was 60°C. Repeated batch hydrolysis of lactose was made with pellets from five Aspergillus strains. A. phoenicis QM 329 showed the least enzyme leakage from the pellets during hydrolysis. From repeated lactose hydrolysis experiments it was estimated that 50% of the mycelia-associated -galactosidase activity remained after 1300 h. Correspondence to: F. Tjerneld     

Human β-endorphin: Development of tolerance and behavioral activity in rats

Human β-endorphin produced a potent antinociceptive response as estimated by the tail-flick test in rats after intraventricular injection. On a molar basis, the peptide was 21 times more potent than morphine and in addition, the peptide produced morphine-like catatonia and hypothermia. These responses were blocked by naloxone. Repeated injections of the peptide induced tolerance to analgesic response, catatonia and hypothermia. Cross tolerance to morphine was also observed.