Decarboxylase Activity Research Articles

Effects of pulsed light on germination and gamma-aminobutyric acid synthesis in brown rice.

This study observed the effects of pulsed light (PL) on the germination and gamma-aminobutyric acid (GABA) production of brown rice and analyzed the correlations among glutamic acid (Glu) content, glutamate decarboxylase (GAD) activity, and GABA content in germinating brown rice. Both germination and GABA content were increased by exposure to PL, and this effect was evident when brown rice was exposed to PL immediately after being soaked. The PL group had significantly higher Glu and GABA content than the control check (CK) group which was unexposed to PL during the germination of brown rice. Glu content peaked at 18 h and GABA peaked at 24h in the PL group, which were 12 h and 6 h earlier than the CK group, respectively. GAD activity of the PL group peaked 12 h after germination, 6 h earlier than the CK group. PL exposure also increased the free amino acid content in the earliest stage of brown-rice germination. During brown-rice germination, the production of GABA is regulated by GAD activity and is significantly positively correlated with Glu content. PL treatment had a significant effect on GAD activity and Glu content during the germination process of brown rice and helped to increase its GABA content. PRACTICAL APPLICATION: This study has shown that pulsed light exposure is an efficient and stable processing method for producing brown rice with high GABA. This will provide a new direction for developing novel germination grain foods.

Decarboxylases as hypothetical targets for actions of organophosphates: Molecular modeling for prediction of hidden and unexpected health threats

The rise of various neurodegenerative disorders are somewhat correlating with the worldwide application of multiple anthropogenic toxicants. Though different possible targets were revealed to date, for example, for organophosphorus compounds (OPs), plenty of questions remain. Several decarboxylases (aromatic amino acid decarboxylase, AADC; histidine decarboxylase, HDC; glutamate decarboxylase, GAD) catalyze the biosynthesis of neurotransmitters and neuromodulators and contain pyridoxal phosphate (PLP) as a cofactor. In the current work, 18 OPs which have different neurotoxicity (chemical warfare agents and pesticides) and can penetrate through the blood–brain barrier, were selected. Then, their possible interaction with these decarboxylases in both apo- and holoforms was revealed using computer modeling methods (molecular docking and dynamics). The main amino acid residues of the enzymes responsible for binding OPs have been identified. Individual substances that are most dangerous from the point of view of a possible negative effect on the activity of several decarboxylases were revealed among studied OPs. Glyphosate should be of special interest, since it is not highly toxic towards serine hydrolases, but may prove to be a strong inhibitor for decarboxylases. Holo-AADC could be the most inhibition-prone enzyme among all those investigated.

A scheme for C4 evolution derived from a comparative analysis of the closely related C3, C3-C4 intermediate, C4-like, and C4 species in the genus Flaveria.

A comparative analysis of the genus Flaveria showed a C4 evolutionary process in which the anatomical and metabolic features of C4 photosynthesis were gradually acquired through C3-C4 intermediate stages. C4 photosynthesis has been acquired in multiple lineages of angiosperms during evolution to suppress photorespiration. Crops that perform C4 photosynthesis exhibit high rates of CO2 assimilation and high grain production even under high-temperature in semiarid environments; therefore, engineering C4 photosynthesis in C3 plants is of great importance in the application field. The genus Flaveria contains a large number of C3, C3-C4 intermediate, C4-like, and C4 species, making it a good model genus to study the evolution of C4 photosynthesis, and these studies indicate the direction for C4 engineering. C4 photosynthesis was acquired gradually through the C3-C4 intermediate stage. First, a two-celled C2 cycle called C2 photosynthesis was acquired by localizing glycine decarboxylase activity in the mitochondria of bundle sheath cells. With the development of two-cell metabolism, anatomical features also changed. Next, the replacement of the two-celled C2 cycle by the two-celled C4 cycle was induced by the acquisition of cell-selective expression in addition to the upregulation of enzymes in the C4 cycle during the C3-C4 intermediate stage. This was supported by an increase in cyclic electron transport activity in response to an increase in the ATP/NADPH demand for metabolism. Suppression of the C3 cycle in mesophyll cells was induced after the functional establishment of the C4 cycle, and optimization of electron transport by suppressing the activity of photosystem II also occurred during the final phase of C4 evolution.

Transcriptome and phytochemical analyses reveal the roles of characteristic metabolites in the taste formation of white tea during the withering process

In the postharvest processing of tea leaves, withering is the first indispensable manufacturing process which produces the mellow, umami and sweet taste of white tea. In this study, we aimed to determine the dynamic changes of the main metabolites and clarify the key differentially expressed genes (DEGs) involved in forming the characteristic taste of white tea during withering. Phytochemical analyses revealed that the contents of total catechins and starch decreased continuously, whereas the contents of theaflavin, γ-aminobutyric acid (GABA), maltose, and soluble sugars increased significantly during withering (from 0–48 h). Meanwhile, the elevation of α-amylase (AMY), β-amylase (BAM), total amylase, and glutamate decarboxylase (GAD) activities may be correlated with the accumulation of GABA and maltose. By transcriptome sequencing, we detected 9 707, 15 921, 17 353, and 17 538 DEGs at 12, 24, 36, and 48 h of the withering process, respectively, compared with 0 h sample (fresh leaves). The transcript levels of most of the DEGs involved in catechin biosynthesis were significantly inhibited, whereas those involved in catechin oxidation were significantly up-regulated, which could be correlated to a decrease in catechin content and an increase in theaflavin content. The DEGs involved in GABA biosynthesis were considerably up-regulated, and the down-regulation of SPMS could reduce the competition for converting spermidine to GABA. The up-regulation of the AMY and BAM genes could trigger starch degradation, resulting in the increase of soluble sugar content. These results provide new insights into the importance of the withering process to the characteristic taste of white tea.

Suppression of cytokine storm and associated inflammatory mediators by salicylaldehyde derivative of pregabalin: An innovative perspective for alleviating airway inflammation and lung remodeling

ObjectivesPregsal is a novel salicylaldehyde derivative of pregabalin, a structural analogue of gamma-aminobutyric acid approved for treating epilepsy, neuropathic pain and diabetic peripheral neuropathy but not trialled to date for airway inflammatory diseases. The current study evaluated a preclinical model of airway inflammation and lung remodelling induced by ovalbumin (OVA) for the potential benefits of pregsal. MethodsWistar rats (N = 10 per group) were divided into four groups sensitized intraperitoneally (i.p.) and then challenged intranasally with OVA, with or without i.p. pregsal (100 mg/kg) or methylprednisolone (MP) (15 mg/kg). Airway inflammation was assessed through inflammatory cell infiltration in the lungs, delayed-type hypersensitivity (DTH), and nitric oxide (NO) level using bronchoalveolar lavage fluid (BALF) and lung tissues. The mRNA expression levels of a panel of inflammatory mediators (cytokines, chemokine and growth factors) in the lungs were measured by the reverse transcription-polymerase chain reaction (RT-PCR). Systemic inflammation was assessed using splenocyte proliferation and total and differential leucocyte count in blood and BALF. Lung remodelling was assessed by wet/dry lung weight ratio, epithelial thickness and goblet cell hyperplasia, hydroxyproline and osteopontin (OPN) levels, arginase activity in lungs, and ornithine decarboxylase (ODC) activity in lung mitochondria. ResultsPregsal significantly alleviated the total and differential leucocyte count in blood and BALF, NO production in BALF and recruitment of inflammatory cells in the lungs. It suppressed the T-cell response and attenuated the OVA-induced lung epithelial thickness, goblet cell hyperplasia, wet/dry lung weight ratio, hydroxyproline and OPN levels, arginase and ODC activity. Levels of inflammatory mediators were also downregulated in the lungs by pregsal. ConclusionsThe key findings of this study indicate that pregsal significantly reduces the development of airway inflammation and lung remodelling by suppression of cytokine storm and associated inflammatory mediators.

Does oxygen affect ageing mechanisms of Pinus densiflora seeds? A matter of cytoplasmic physical state.

During desiccation, the cytoplasm of orthodox seeds solidifies into an intracellular glass with highly restricted diffusion and molecular mobility. Temperature and water content govern seed ageing rates, while oxygen (O2) can promote deteriorative reactions. However, whether the cytoplasmic physical state affects involvement of O2 in seed ageing remains unresolved. We aged Pinus densiflora seeds by controlled deterioration (CD) at 45 °C and distinct relative humidity (RH), resulting in cells with a glassy (11% and 30% RH) or fluid (60% and 80% RH) cytoplasm. Hypoxic conditions (0.4% O2) during CD delayed seed deterioration, lipid peroxidation, and decline of antioxidants (glutathione, α-tocopherol, and γ-tocopherol), but only when the cytoplasm was glassy. In contrast, when the cytoplasm was fluid, seeds deteriorated at the same rate regardless of O2 availability, while being associated with limited lipid peroxidation, detoxification of lipid peroxide products, substantial loss of glutathione, and resumption of glutathione synthesis. Changes in metabolite profiles provided evidence of other O2-independent enzymatic reactions in a fluid cytoplasm, including aldo-keto reductase and glutamate decarboxylase activities. Biochemical profiles of seeds stored under seed bank conditions resembled those obtained after CD regimes that maintained a glassy cytoplasm. Overall, O2 contributed more to seed ageing when the cytoplasm was glassy, rather than fluid.

Advances in understanding the enzymatic potential and production of ochratoxin A of filamentous fungi isolated from cocoa fermented beans

In this work, we determined the degree of filamentous fungi contamination in cocoa fermented seeds and the fungal potential to produce enzymes that could contribute to the cocoa quality. Internal transcribed spacer amplicon sequencing (ITS) showed 11 different species with Byssochlamys spectabilis, Aspergillus niger, Aspergillus flavus, Aspergillus carbonarius, Circinella muscae and Penicillium citrinum as the most abundant species. Our results evidenced intra and inter-species differences in the enzymes production. Cellulases, β-glucosidase, ornithine decarboxylase and phenylalanine decarboxylase were the most diffused enzymes expressed in the 53 strains here studied. Moreover, A. niger (6/12), A. carbonarius (2/3), and P. citrinum (3/3) showed high pectinolytic activity. Remarkable was the amino decarboxylase activity of P. citrinum, and A. flavus strains. For the first time we reported the presence of B. spectabilis in cocoa fermented beans, which could play an important role in the biogenic amines formation. In addition, we explored the capability of the Aspergillus section Nigri strains, to produce ochratoxin A (OTA) in a cacao model system (CPMS) and in malt extract medium (MEL). We observed that CPMS, but not MEL, stimulated the OTA production in 6 out 15 strains of Aspergillus section Nigri, reaching values ranging between 1.70 and 4995 μg OTA kg−1 dry mycelium.

A Tear Metabolomic Profile Showing Increased Ornithine Decarboxylase Activity and Spermine Synthesis in Thyroid-Associated Orbitopathy.

About half of patients with Graves’ disease develop an orbitopathy related to an inflammatory expansion of the periorbital adipose tissue and muscles. We used a targeted metabolomic approach measuring 188 metabolites by mass spectrometry to compare the metabolic composition of tears in patients with active (n = 21) versus inactive (n = 24) thyroid-associated orbitopathy. Among the 44 metabolites accurately measured, 8 showed a significant alteration of their concentrations between the two groups. Two short-chain acylcarnitines, propionylcarnitine and butyrylcarnitine, and spermine showed increased concentrations in the tears of patients with active orbitopathy, whereas ornithine, glycine, serine, citrulline and histidine showed decreased concentrations in this group. In addition, the ratio putrescine/ornithine, representing the activity of ornithine decarboxylase, was significantly increased in patients with active compared to inactive orbitopathy (p = 0.0011, fold change 3.75). The specificity of this candidate biomarker was maintained when compared to a control group with unclassified dry eye disease. Our results suggest that the stimulation of ornithine decarboxylase by TSH receptor autoantibodies in orbital fibroblasts could lead to increased synthesis of spermine, through the increased activity of ornithine decarboxylase, that may contribute to periorbital expansion in Graves’ ophthalmopathy.

Characterizing serotonin biosynthesis in Setaria viridis leaves and its effect on aphids

A combined transcriptomic and metabolic analysis of Setaria viridis leaves responding to aphid infestation was used to identify genes related to serotonin biosynthesis. Setaria viridis (green foxtail), a short life-cycle C4 plant in the Poaceae family, is the wild ancestor of Setaria italica (foxtail millet), a resilient crop that provides good yields in dry and marginal land. Although S. viridis has been studied extensively in the last decade, the molecular mechanisms of insect resistance in this species remain under-investigated. To address this issue, we performed a metabolic analysis of S. viridis and discovered that these plants accumulate the tryptophan-derived compounds tryptamine and serotonin. To elucidate the defensive functions of serotonin, Rhophalosiphum padi (bird cherry-oat aphids) were exposed to this compound, either by exogenous application to the plant medium or with artificial diet bioassays. In both cases, exposure to serotonin increased aphid mortality. To identify genes that are involved in serotonin biosynthesis, we conducted a transcriptome analysis and identified several predicted S. viridis tryptophan decarboxylase (TDC) and tryptamine 5-hydroxylase (T5H) genes. Two candidate genes were ectopically expressed in Nicotiana tabacum, where SvTDC1 (Sevir.6G066200) had tryptophan decarboxylase activity, and SvT5H1 (Sevir.8G219600) had tryptamine hydroxylase activity. Moreover, the function of the SvTDC1 gene was validated using virus-induced gene silencing in S. italica, which caused a reduction in serotonin levels. This study provides the first evidence of serotonin biosynthesis in Setaria leaves. The biosynthesis of serotonin may play an important role in defense responses and could prove to be useful for developing more pest-tolerant Setaria italica cultivars.

Changes in the free amino acid profile of pickling cucumber during lactic acid fermentation.

Free amino acid (FAA) profiles of fresh, acidified, naturally fermented, and starter culture fermented cucumbers were analyzed by liquid chromatography triple quadrupole mass spectrometry. Fermented cucumbers contained more total FAA than acidified cucumbers (1,302 ± 102mg/kg and 635 ± 35mg/kg, respectively). Total FAA content of fermented cucumber was similar regardless of brine salt levels (2-6% NaCl) and starter culture addition. Glutamine (1491.4± 69.3mg/kg), γ-aminobutyric acid (GABA, 269.6± 21.4mg/kg), asparagine (113.0± 6.4mg/kg), and citrulline (110.3±8.5mg/kg) were the most abundant FAA in fresh pickling cucumber, whereas GABA (181.3±21.5mg/kg), isoleucine (165.2± 11.2mg/kg), leucine (129.8± 10.9mg/kg), and lysine (110.9± 5.0mg/kg) were the most abundant in fermented cucumber. GABA and ornithine were produced during fermentation, indicating glutamate decarboxylase and arginine deiminase activities. Notably, ornithine was significantly higher in natural (63.3±31.5mg/kg) versus starter culture fermented cucumbers (3.0±0.7mg/kg). This new information on FAA composition of fresh and fermented pickling cucumbers shows the impact of fermentation conditions on cucumber amino acid profiles while providing insight for manipulating fermentations for health promotion and consumer acceptance. PRACTICAL APPLICATION: This study reports changes in the free amino acid profiles of raw, fermented and acidified cucumbers, which may be valuable for understanding the impact of these foods on human health and nutrition. This information is useful for food microbiologists studying the metabolism of lactic acid bacteria during fermentation and/or designing starter cultures and could contribute to the development of novel fermented cucumber pickle products with enhanced nutritional value.

Melatonin and KNO3 Application Improves Growth, Physiological and Biochemical Characteristics of Maize Seedlings under Waterlogging Stress Conditions.

Simple SummaryWaterlogging is one of the serious abiotic stresses that retards plant growth and reduces crop productivity. Therefore, exploring effective ways for alleviating the impacts of waterlogging stress has important theoretical and practical applications. Melatonin is a pleiotropic molecule that influences many diverse actions to enhance plant growth. Therefore, investigating efficient waterlogging mitigation measures has both theoretical and practical significance. The objectives of the present research were to examine the efficiency of melatonin and KNO3 seed soaking and foliar application on alleviating the waterlogging inhibited growth performance of maize seedlings. The results showed that melatonin and KNO3 significantly improved the plant growth and biochemical parameters of maize seedlings under waterlogging stress conditions. Overall, the application of 100 µM melatonin with 0.50 g KNO3 via seed soaking and foliar spray can be used as a potential mitigating strategy for improving the growth of maize seedlings and alleviating the ill effects of waterlogging stress.Waterlogging is one of the serious abiotic stresses that inhibits crop growth and reduces productivity. Therefore, investigating efficient waterlogging mitigation measures has both theoretical and practical significance. The objectives of the present research were to examine the efficiency of melatonin and KNO3 seed soaking and foliar application on alleviating the waterlogging inhibited growth performance of maize seedlings. In this study, 100 µM melatonin and different levels (0.25, 0.50 and 0.75 g) of potassium nitrate (KNO3) were used in seed soaking and foliar applications. For foliar application, treatments were applied at the 7th leaf stage one week after the imposition of waterlogging stress. The results showed that melatonin with KNO3 significantly improved the plant growth and biochemical parameters of maize seedlings under waterlogging stress conditions. However, the application of melatonin with KNO3 treatments increased plant growth characteristics, chlorophyll content, and the net photosynthetic rate at a variable rate under waterlogging stress. Furthermore, melatonin with KNO3 treatments significantly reduced the accumulation of hydrogen peroxide (H2O2) and malondialdehyde (MDA), and it decreased the activity of pyruvate decarboxylase and alcohol dehydrogenase, while increasing enzymatic activities and soluble protein content of maize seedlings under waterlogging stress conditions. Overall, our results indicated that seed soaking with 100 µM melatonin and 0.50 g KNO3 was the most effective treatment that significantly improved the plant growth characteristics, chlorophyll content, photosynthetic rate, and enzymatic activity of maize seedling under waterlogging conditions.

Enrichment of γ-aminobutyric acid in mulberry leaves and the inhibitory effects of the water extract on ACE and α-glucosidase activity

To improve the functional properties of mulberry leaves, different methods were used to treat harvested mulberry leaves to accumulate γ-aminobutyric acid (GABA). The results showed that soaking, cold shock, and anoxic treatments all significantly increased GABA content in mulberry leaves, which were all closely related to the increase of glutamate decarboxylase (GAD) activity and Ca2+ content. Further studies showed that the water extract of the treated mulberry leaves had a higher angiotensin I-converting enzyme (ACE) inhibitory activity than that of the untreated sample, which was close to the effects of using the same amount of GABA alone. The result of molecular docking showed that GABA was stretched into the hydrophobic pocket of ACE to form a stable hydrophobic binding with Ala-354 and Val-380 residues, and formed the hydrogen bond interaction with the residue Glu-384. Importantly, GABA could directly chelated with the zinc ion (bond lengths: 2.9 and 3.1 Å), which was the main interaction between the GABA and ACE. However, the inhibition of mulberry leaf extract on α-glucosidase was not closely related to the GABA content. The results showed that the α-glucosidase inhibitory activity was significantly increased by treating with 6 g/L sodium glutamate for 15 h (IC50 =0.29 mg/mL), and further LC/MS analysis demonstrated that some active compounds especially neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid and rutin were significantly increased in the extract. These data indicated that GABA enrichment was an effective postharvest treatment of mulberry leaves, which can improve its prospects as functional raw materials for industrial production.

Precise measurement of decarboxylase and applied cascade enzyme for simultaneous cadaverine production with carbon dioxide recovery

BackgroundBio-based nylon is synthesized from putrescine, cadaverine, or 4-amino butyric acid through decarboxylases by a green process aimed toward a renewable nylon market. Thus, quantifying the activity of the key enzyme decarboxylase with precision and efficiency, is critical for the development of the system. Moreover, one molecular carbon dioxide releasing from the reaction should be considered in greenhouse gas effect. MethodsWe established a new assay based on pH value decline by using different alkaline solution to absorb and capture the CO2 released during decarboxylation. The pH change is corresponding to decarboxylase activity. On the other hand, carbonic anhydrase (CA), phosphoribulokinase (PRK) and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo) genes involved in CO2 respiration, were applied with lysine decarboxylase (CadA) for simultaneous production of cadaverine and recovery of CO2. Significant FindingsThe correlation between lysine, glutamate, or arginine conversion and its pH changes in different decarboxylases was successfully determined using a numerical equation with a high regression value (R2 > 0.95). For cascade enzymatic reaction, RuBisCo was hardly co-expressed with CadA. Therefore, the strain with CadA and CA resulted in the release of the least amount of CO2 among all the varieties, reaching the best CO2 assimilation capability of −4.95 g-CO2/g-dry cell weight (DCW) and an acceptable DAP of 15.67 g/L. This illustrates a new trend of bio-mitigation of CO2 with simultaneous production of a variety of desirable chemicals.

Porphyria Cutanea Tarda in a 54-Year-Old Patient with a History of Hepatitis C: A Case Report

Background Porphyria CutaneaTarda (PCT) is the most common type of porphyria and is caused by a decrease in the activity of the hepatic enzyme uroporphyrinogen decarboxylase. It is expressed in both a sporadic form and genetic form and typically presents with cutaneous manifestations described as skin blisters in sun exposed areas. Case A 54-year-old male presented complaining of bullous itchy lesions on his hands and upper extremities that were at different stages of healing. Lab results were consistent with porphyria including elevated serum total porphyrins. He was scheduled for phlebotomy every other week for six weeks, hydroxychloroquine, minimize any sun exposure and to completely stop smoking. Conclusion Widespread skin lesions associated with underlying liver disease is a characteristic presentation for PCT. Hepatitis C is an antecedent risk factor for PCT, but can now be treated with antiviral therapy with the expectation of attainment of a sustained virologic response. Improvements in arresting progressive liver disease in Hepatitis C patients may improve PCT symptoms, as well. Keywords: Porphyria CutaneaTarda; Hepatitis C; Acquired liver disease.

Postharvest γ-aminobutyric acid application mitigates chilling injury of aonla (Emblica officinalis Gaertn.) fruit during low temperature storage

Aonla is a nutrients rich underutilized subtropical fruit. It has short shelf life and is prone to chilling injury (CI) below 6 °C. So, the effect of γ-aminobutyric acid [5 mmol L−1 (GABA)] was investigated on aonla fruit CI mitigation during low temperature storage at 5 ± 1 °C for 24 d. Results showed that GABA treatment markedly suppressed CI, decay incidence, membrane permeability, malondialdehyde, superoxide anion and hydrogen peroxide in contrast with control. Treated fruit showed higher activity of glutamate decarboxylase (GAD) and GABA transaminase (GABA-T) enzymes which ultimately resulted in increased endogenous GABA content accumulation. Similarly, GABA application stimulated the activities of Δ1-pyrroline-5-carboxylate synthetase (P5CS) and ornithine aminotransferase (OAT) enzymes which thus enhanced proline accumulation. The GABA treated fruit exhibited higher phenylalanine ammonia lyase (PAL) and reduced polyphenol oxidase (PPO) enzyme activity and showed higher total phenols accumulation. In addition, total flavonoids, ascorbic acid and glutathione content were also significantly higher in GABA treated aonla fruit. In addition, GABA treated aonla fruit exhibited markedly higher ascorbate peroxidase (APX) catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) activities in comparison with the control. In addition, GABA treated fruit showed lower total soluble solids, ripening index and juice pH along with markedly higher concentration of titratable acidity. In conclusion, GABA treatment could be used for CI mitigation and overall quality maintenance of cold stored aonla fruit.

Inhibitory effects of nicotinic acid, glycine, and other food additives on biogenic amine formation in Baechu kimchi fermentation

The current study was performed to reduce biogenic amine (BA) content in Baechu (napa cabbage) kimchi using food additives for prevention of BA-related risks. Through in-vitro experiments, among 9 selected additives, nicotinic acid and glycine exhibited the highest inhibitory effects on the tyramine production of a prolific tyramine-producing Levilactobacillus brevis BC1M20, showing inhibition of up to 100% and 78%, respectively, depending on the concentrations tested, compared to the control. In Baechu kimchi fermentation experiments, tyramine content in kimchi prepared with L. brevis BC1M20 and either 1% nicotinic acid or 5% glycine was dramatically reduced by 90.66% and 82.51%, respectively, compared to naturally fermented kimchi. When compared to kimchi prepared with the inoculum and no food additives, remarkable tyramine reductions of 87.28% and 76.20% were achieved by 1% nicotinic acid and 5% glycine, respectively. With the additives, content of 3–5 other BAs was also considerably reduced by 22.57%–76.64%. Such reductions were likely attributed to the inhibition of growth and/or amino acid decarboxylase activity of BA-producing bacteria. Consequently, the findings of this study can be valuable in the prevention of BA-related food safety issues in kimchi.

Improving the Stability and Activity of Arginine Decarboxylase at Alkaline pH for the Production of Agmatine

Agmatine, involved in various modulatory actions in cellular mechanisms, is produced from arginine (Arg) by decarboxylation reaction using arginine decarboxylase (ADC, EC 4.1.1.19). The major obstacle of using wild-type Escherichia coli ADC (ADCes) in agmatine production is its sharp activity loss and instability at alkaline pH. Here, to overcome this problem, a new disulfide bond was rationally introduced in the decameric interface region of the enzyme. Among the mutants generated, W16C/D43C increased both thermostability and activity. The half-life (T1/2) of W16C/D43C at pH 8.0 and 60°C was 560 min, which was 280-fold longer than that of the wild-type, and the specific activity at pH 8.0 also increased 2.1-fold. Site-saturation mutagenesis was subsequently performed at the active site residues of ADCes using the disulfide-bond mutant (W16C/D43C) as a template. The best variant W16C/D43C/I258A displayed a 4.4-fold increase in the catalytic efficiency when compared with the wild-type. The final mutant (W16C/D43C/I258A) was successfully applied to in vitro synthesis of agmatine with an improved yield and productivity (>89.0% yield based on 100 mM of Arg within 5 h).

Malonyl-acyl carrier protein decarboxylase activity promotes fatty acid and cell envelope biosynthesis in Proteobacteria

Bacterial fatty acid synthesis in Escherichia coli is initiated by the condensation of an acetyl-CoA with a malonyl-acyl carrier protein (ACP) by the β-ketoacyl-ACP synthase III enzyme, FabH. E. coli ΔfabH knockout strains are viable because of the yiiD gene that allows FabH-independent fatty acid synthesis initiation. However, the molecular function of the yiiD gene product is not known. Here, we show the yiiD gene product is a malonyl-ACP decarboxylase (MadA). MadA has two independently folded domains: an amino-terminal N-acetyl transferase (GNAT) domain (MadAN) and a carboxy-terminal hot dog dimerization domain (MadAC) that encodes the malonyl-ACP decarboxylase function. Members of the proteobacterial Mad protein family are either two domain MadA (GNAT-hot dog) or standalone MadB (hot dog) decarboxylases. Using structure-guided, site-directed mutagenesis of MadB from Shewanella oneidensis, we identified Asn45 on a conserved catalytic loop as critical for decarboxylase activity. We also found that MadA, MadAC, or MadB expression all restored normal cell size and growth rates to an E. coli ΔfabH strain, whereas the expression of MadAN did not. Finally, we verified that GlmU, a bifunctional glucosamine-1-phosphate N-acetyl transferase/N-acetyl-glucosamine-1-phosphate uridylyltransferase that synthesizes the key intermediate UDP-GlcNAc, is an ACP binding protein. Acetyl-ACP is the preferred glucosamine-1-phosphate N-acetyl transferase/N-acetyl-glucosamine-1-phosphate uridylyltransferase substrate, in addition to being the substrate for the elongation-condensing enzymes FabB and FabF. Thus, we conclude that the Mad family of malonyl-ACP decarboxylases supplies acetyl-ACP to support the initiation of fatty acid, lipopolysaccharide, peptidoglycan, and enterobacterial common antigen biosynthesis in Proteobacteria.

Technological and Safety Characterization of Kocuria rhizophila Isolates From Traditional Ethnic Dry-Cured Ham of Nuodeng, Southwest China.

Nuodeng ham is known for its unique processing techniques and flavor. In the present study, proteolytic microorganisms from cured artisanal Nuodeng ham were investigated in order to identify and select potential starter cultures for its faster and safer fermentation. Eight isolates, accounting for 57% of proteolytic microorganisms, were found to be related to Kocuria rhizophila. Relevant properties of K. rhizophila as potential starter culture were evaluated in vitro for the first time. Intra-species diversities were found in phylogenetic and physiological properties of K. rhizophila isolates. Nevertheless, desirable attributes, such as halo-tolerance, nitrate reductase and protease activity, as well as the absence of antimicrobial resistance and amino acid decarboxylase activity, were observed in selected isolates. Moreover, genome analysis of isolates K24 and K45 confirmed their lack of typical genes for virulence, antimicrobial resistance and amino acid decarboxylase. K. rhizophila may thus represent a novel starter candidate of coagulase-negative cocci group and contribute to color and flavor development of fermented meats.

Contribution of pre-storage melatonin application to chilling tolerance of some mango fruit cultivars and relationship with polyamines metabolism and γ-aminobutyric acid shunt pathway

The efficacy of pre-storage application of 100 µM melatonin on conferring chilling tolerance in fruit of four mango cultivars stored at 5 ± 1 ºC for 28 d was investigated. ‘Langra’ and ‘Gulab Jamun’ mangoes had maximum and minimum chilling tolerance, respectively. Accumulation of endogenous polyamines and γ-aminobutyric acid (GABA) in the peel and the pulp of ‘Langra’ fruit treated with melatonin was a possible reason for its chilling tolerance. Accumulation of endogenous polyamines was associated with higher arginine decarboxylase (ADC) and ornithine decarboxylase (ODC), and lower diamine oxidase (DAO) and polyamine oxidase (PAO) activities in the peel and pulp. Additionally, higher GABA shunt (Glutamate decarboxylase and GABA-transaminase) activity was associated with elevated GABA content in ‘Langra’ mangoes. The limited effectiveness of melatonin treatment for chilling injury alleviation in ‘Chaunsa’ was probably due to higher polyamines accumulation accompanied with lower PAO and DAO activity in the peel and pulp of the fruit, in addition to higher ADC and ODC in the peel.