EPR evidence of two structurally different diferric sites in Mycobacterium tuberculosis R2-2 ribonucleotide reductase protein

Mixed-valent species were generated in the diiron site of active (with tyrosyl free radical) and met (without radical) forms of protein R2-2 in a class Ib ribonucleotide reductase from Mycobacterium tuberculosis by low temperature reduction (γ-irradiation) at 77 K. The primary mixed-valent EPR signal is a mixture of two components with axial symmetry and g_av<2.0, observable at temperatures up to 77 K, and assigned to antiferromagnetically coupled high spin ferric/ferrous sites. The two components in the primary EPR signal can be explained by the existence of two structurally distinct μ-oxo-bridged diferric centers, possibly related to structural heterogeneity around the iron site, and/or different properties of the two polypeptide chains in the homodimeric protein after the radical reconstitution reaction. Annealing of the irradiated R2-2 samples to 143 K transforms the primary EPR signal into a rhombic spectrum characterized by g_av<1.8 and observable only below 25 K. This spectrum is assigned to a partially relaxed form with a μ-hydroxo-bridge. Further annealing at 228 K produces a new complex rhombic EPR spectrum composed of at least two components. An identical EPR spectrum was observed and found to be stable upon chemical reduction of Mycobacterium tuberculosis RNR R2-2 at 293 K by dithionite.     

Effects of sodium on mineral nutrition in rose plants

The effects of sodium (Na⁺) ion concentration on shoot elongation, uptake of ammonium (NH₄⁺) and nitrate (NO₃^?) and the activities of nitrate reductase (NR) and glutamine synthetase (GS) were studied in rose plants (Rosa hybrida cv. “Lambada”). The results showed that shoot elongation was negatively correlated with sodium concentration, although no external symptoms of toxicity were observed. Nitrate uptake decreased at high sodium levels, specifically at 30 meq litre4 of sodium. As flower development was normal under high saline conditions, this could suggest that nitrogen was being mobilised from shoot and leaf reserves. Ammonium uptake was not affected by any of the salt treatments applied probably because it diffuses through the cell membrane at low concentrations. Nitrate reductase activity was reduced by 50% at 30 meq litre 1 compared with control treatment, probably due to a decrease in the free nitrate related to nitrate uptake pattern. None of the salt treatments used affected total leaf GS activity (both chloroplastic and cytosolic isoforms) or leaf NPK mineral contents. Nitrate reductase activity in leaves increased at 10 meq litre^?1 of sodium and GS activity in roots (cytosolic isoform only) followed the same pattern as NR. It is suggested that the activation of both enzymes at low salt level could be attributed to the beneficial effect of increased sulphur in the nutrient solutions.     

Improving the Mineral Nutrition in Grafted Watermelon Plants: Nitrogen Metabolism

Watermelon (Citrullus lanatus [Trumb.] Mansfeld cv. Early Star), was used as scion grafted onto three cultivars of pumpkin (Cucurbita pepo L. cvs. Brava, Shintoza and Kamel) used as rootstocks and ungrafted Early Star plants were used as control. The rootstocks showed a high capacity for N uptake and transport to the scion where N reduction and assimilation improved growth of the scion in grafted plants with respect to the control.     

COMPARISONS OF NITRATE UPTAKE, STORAGE, AND REDUCTION IN MARINE DIATOMS AND FLAGELLATES

Diatoms, but not flagellates, have been shown to increase rates of nitrogen release after a shift from a low growth irradiance to a much higher experimental irradiance. We compared NO₃ ^? uptake kinetics, internal inorganic nitrogen storage, and the temperature dependence of the NO₃ ^? reduction enzymes, nitrate (NR) and nitrite reductase (NiR), in nitrogen‐replete cultures of 3 diatoms (Chaetoceros sp., Skeletonema costatum, Thalassiosira weissflogii) and 3 flagellates (Dunaliella tertiolecta, Pavlova lutheri, Prorocentrum minimum) to provide insight into the differences in nitrogen release patterns observed between these species. At NO₃ ^? concentrations <40 μmol‐N·L ^{? 1}, all the diatom species and the dinoflagellate P. minimum exhibited saturating kinetics, whereas the other flagellates, D. tertiolecta and P. lutheri, did not saturate, leading to very high estimated K _s values. Above ～60 μmol‐N·L ^{? 1}, NO₃ ^? uptake rates of all species tested continued to increase in a linear fashion. Rates of NO₃ ^? uptake at 40 μmol‐N·L ^{? 1}, normalized to cellular nitrogen, carbon, cell number, and surface area, were generally greater for diatoms than flagellates. Diatoms stored significant amounts of NO₃ ^? internally, whereas the flagellate species stored significant amounts of NH₄ ⁺ . Half‐saturation concentrations for NR and NiR were similar between all species, but diatoms had significantly lower temperature optima for NR and NiR than did the flagellates tested in most cases. Relative to calculated biosynthetic demands, diatoms were found to have greater NO₃ ^? uptake and NO₃ ^? reduction rates than flagellates. This enhanced capacity for NO₃ ^? uptake and reduction along with the lower optimum temperature for enzyme activity could explain differences in nitrogen release patterns between diatoms and flagellates after an increase in irradiance.     

高温对两种卡帕藻的酶活性、色素含量与叶绿素荧光的影响

在室内采用静置培养法,以25℃为对照,研究高温(32℃、35℃和40℃)对长心卡帕藻Kappaphycus alvarezii与异枝卡帕藻K.striatum的硝酸还原酶(NR)和过氧化物酶(POD)活性、叶绿素a和藻红蛋白含量的影响,并采用调制叶绿素荧光技术,测定高温处理后2种藻的叶绿素荧光参数变化趋势.结果表明:高温对2种卡帕藻的NR和POD活性有显著影响.二者的NR活性均在35℃时最低,32℃时最高;异枝卡帕藻的POD活性在35℃时最高,而长心卡帕藻的在35℃时最低.在25-40℃之间,随着温度升高,异枝卡帕藻的叶绿素a含量逐渐减少,藻红蛋白含量先降后升,35℃时最高,而长心卡帕藻的两种色素含量皆在25℃时最高.在32-40℃条件下,随着温度升高,2种藻的实际光合效率(Y)和相对光合电子传递速率(ETR)明显下降,温度越高,下降程度越大;短期高温可刺激2种卡帕藻的光合活性增强,随着处理时间延长,异枝卡帕藻的光合活性下降,但差异不显著,而长心卡帕藻的光合活性显著下降.综合上述指标,显示32℃以上高温对2种卡帕藻产生胁迫,异枝卡帕藻对热胁迫的耐受力明显强于长心卡帕藻.     

响应面法优化海洋镰刀腐皮菌产出HMG-CoA还原酶抑制剂的培养条件

【目的】采用响应面法对海洋微生物镰刀腐皮菌FG319发酵产生MFS(Metabolite of Fusarium solani FG319)培养条件进行优化。【方法】在单因素试验结果的基础上,依据Box-Behnken中心组合原则设计诱导物添加量、培养时间、培养温度的3因素3水平响应面实验,以MFS产出量为响应值优化镰刀腐皮菌FG319的培养条件。【结果】镰刀腐皮菌FG319最优培养条件为诱导物添加量0.6%、培养时间7 d、培养温度22°C,在此培养条件下,MFS最高产量达到20.11 mg/L,是优化前的4倍,与理论预测的相对误差为0.64%,实测值与响应面预测值拟合良好。镰刀腐皮菌FG319代谢产物MFS在HMG-CoA还原酶和NADPH构筑的分子评价反应体系,当MFS添加到100 mg/L时,具有类似洛伐他汀抑制HMG-CoA还原酶的最大抑制率。【结论】响应面试验设计对镰刀腐皮菌FG319培养条件的优化是有效的,其次生代谢产物MFS体外抑制HMG-CoA还原酶的效果也是明显的。     

Cryo-EM structure of the fatty acid reductase LuxC–LuxE complex provides insights into bacterial bioluminescence

The discovery of reduced flavin mononucleotide and fatty aldehydes as essential factors of light emission facilitated study of bacterial luminescence. Although the molecular mechanisms underlying bacterial luminescence have been studied for more than 60 years, the structure of the bacterial fatty acid reductase complex remains unclear. Here, we report the cryo-EM structure of the Photobacterium phosphoreum fatty acid reductase complex LuxC–LuxE to a resolution of 2.79 Å. We show that the active site Lys238/Arg355 pair of LuxE is >30 Å from the active site Cys296 of LuxC, implying that catalysis relies on a large conformational change. Furthermore, mutagenesis and biochemical experiments support that the L-shaped cleft inside LuxC plays an important role in substrate binding and reaction. We obtained a series of mutants with significantly improved activity as measured by in vitro bioluminescence assays and demonstrated that the double mutant W111A/F483K displayed the highest activity (370% of the WT). Our results indicated that the activity of LuxC significantly affects the bacterial bioluminescence reaction. Finally, we expressed this mutated lux operon in Escherichia coli but observed that the in vivo concentrations of ATP and NADPH limited the enzyme activity; thus, we conclude that the luminous intensity mainly depends on the level of metabolic energy.     

微生物亚硝酸盐还原酶的研究进展

亚硝酸盐还原酶(Nitrite reductase,简称NiR,EC1.7.2.1)是催化亚硝酸盐(Nitrite,简称NIT)还原的一类酶,可降解NIT为NO或NH3,是自然界氮循环过程的关键酶。本文详细阐述亚硝酸盐还原酶的分类、结构特点、催化机制以及现阶段的应用领域,为深入研究亚硝酸还原酶提供参考。     

炭疽菌不利用硝酸盐突变体的筛选及鉴定

将从杉木和大叶黄杨上分离获得的８个胶孢炭疽菌（Colletoctrichum gloeosporioides）分离物培养在含氯酸钾的平板上,得到快速生长抗氯酸钾的不利用硝酸盐的突变体(Nit)。所有的突变体经鉴定分属于３种表现型,即硝酸还原酶结构位点（nit１）,硝酸盐同化途径的专化调节位点（nit３）,和钼辅因位点（nitM）。分离物发生突变的频率随氯酸钾浓度的增加而提高,并且不同的氮源在一定程度上会影响突变表型种类。除ＣＣ３外,所有的分离物都是自身亲和的,即不同表型的突变体能遗传互补,其     

Comparative study of the antioxidant activity of the essential oils of five plants against the H2O2 induced stress in Saccharomyces cerevisiae

The purpose of this work was to investigate the protective effect of five essential oils (EOs); Rosmarinus officinalis, Thymus vulgaris, Origanum compactum Benth., Eucalyptus globulus Labill. and Ocimum basilicum L.; against oxidative stress induced by hydrogen peroxide in Saccharomyces cerevisiae. The chemical composition of the EOs was analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC/MS). The in vitro antioxidant activity was evaluated and the protective effect of EOs was investigated. Yeast cells were pretreated with different concentrations of EOs (6.25–25 µg/ml) for an hour then incubated with H₂O₂ (2 mM) for an additional hour. Cell viability, antioxidants (Catalase, Superoxide dismutase and Glutathione reductase) and metabolic (Succinate dehydrogenase) enzymes, as well as the level of lipid peroxidation (LPO) and protein carbonyl content (PCO) were evaluated. The chemical composition of EOs has shown the difference qualitatively and quantitatively. Indeed, O. compactum mainly contained Carvacrol, O. basilicum was mainly composed of Linalool, T. vulgaris was rich in thymol, R. officinalis had high α-Pinene amount and for E. globulus, eucalyptol was the major compound. The EOs of basil, oregano and thyme were found to possess the highest amount of total phenolic compounds. Moreover, they have shown the best protective effect on yeast cells against oxidative stress induced by H₂O₂. In addition, in a dose dependent manner of EOs in yeast medium, treated cells had lower levels of LPO, lower antioxidant and metabolic enzymes activity than cells exposed to H₂O₂ only. The cell viability was also improved. It seems that the studied EOs are efficient natural antioxidants, which can be exploited to protect against damages and serious diseases related to oxidative stress.