Under a suitable condition of crystallization, dark brown short rhombohedron crystals could be obtained from FeMoco-deficient MoFe protein (DeltanifE Avl) purified from a nifE deleted mutant DJ35 of Azotobacter vinela...Under a suitable condition of crystallization, dark brown short rhombohedron crystals could be obtained from FeMoco-deficient MoFe protein (DeltanifE Avl) purified from a nifE deleted mutant DJ35 of Azotobacter vinelandii Lipmann grown in NH3-limited medium. The number, size and quality of crystals were significantly affected by either the concentration of precipitants and buffer or diffusion method. The longest sides of the largest crystal of DeltanifE Avl protein, which was obtained by vapor diffusion in the hanging drop method, were 0.12 and 0.13 mm, respectively.展开更多
Under a suitable condition of crystallization, dark brown short rhombohedron crystals could be obtained from nitrogenase MnFe protein purified from a mutant UW3 of Azotobacter vinelandii Lipmann grown in Mn-containing...Under a suitable condition of crystallization, dark brown short rhombohedron crystals could be obtained from nitrogenase MnFe protein purified from a mutant UW3 of Azotobacter vinelandii Lipmann grown in Mn-containing but Mo- and NH3-free medium. The possibility of crystallization, and number, size and quality of crystals were obviously dependent on concentrations of NaCl, MgCl2, PEG 8000,Tris and Hepes buffer and on methods for crystallization. PEG concentration affected on the shape of the crystals. The optimal, concentrations of the chemicals for crystallization of MnFe protein were slightly different from those for crystallization of Delta nifZ MoFe protein from a nifZ deleted strain of Azotobacter vinelandii. SDS-PAGE showed that the protein from the dissolved crystals was almost the same as MnFe protein before crystallization, indicating that the crystal was formed from MnFe protein.展开更多
A mutant UW 3, which is unable to fix N 2 in the presence of Mo (Nif -) but undergo phenotypic reversal to Nif + under Mo deficiency, was able to grow in Mo- and NH 3-deficient medium containing Mn, and the growt...A mutant UW 3, which is unable to fix N 2 in the presence of Mo (Nif -) but undergo phenotypic reversal to Nif + under Mo deficiency, was able to grow in Mo- and NH 3-deficient medium containing Mn, and the growth was accelerated by Mn at low concentration. A partly purified nitrogenase component Ⅰ protein separated from UW 3 grown in the Mn-containing medium was shown to contain Fe and Mn atoms (ratio of Fe/Mo/Mn: 10.41/0.19/1.00) with C 2H 2- and H +-reducing activity which almost equal to half of that of MoFe protein purified from wild-type mutant of Azotobacter vinelandii Lipmann. This protein was obviously different from MoFe protein in both absorption spectrum and circular dichroism, and the molecular weight of subunits in Mn-containing protein was close to that of α subunit in MoFe protein. The preliminary results indicated that the protein containing Mn might be a nitrogenase component Ⅰ protein.展开更多
A mutant UW 3, which is unable to fix N 2 in the presence of Mo (Nif -) but can undergo phenotypic reversal to Nif + under Mo_deficient conditions, was able to grow in Cr_containing but Mo_ and NH 3_deficient...A mutant UW 3, which is unable to fix N 2 in the presence of Mo (Nif -) but can undergo phenotypic reversal to Nif + under Mo_deficient conditions, was able to grow in Cr_containing but Mo_ and NH 3_deficient medium. A partly purified nitrogenase component Ⅰ protein obtained from UW 3 grown on the Cr_containing medium was shown to contain Fe and Cr (atom ratio of Fe to Cr and Mo to Cr: 11.60 and 0.41) and to have 70% of the C 2H 2_ and H +_reduction activity of MoFe protein from the wild_type strain of Azotobacter vinelandii Lipmann. The Cr_containing protein was different in subunit composition from that of MnFe protein purified from the mutant strain grown in the presence of Mn, but similar to that of MoFe protein, that is, it was a tetramer composed of two different subunits (α 2β 2). The preliminary results indicated that the Cr_containing protein might be a nitrogenase component Ⅰ protein.展开更多
Nitrogenase CrFe protein and MnFe protein were purified from a mutant strain UW3 of Azotobacter vinelandii Lipmann grown on a medium containing Cr and Mn, respectively. In order to meet the requirement for crystal gro...Nitrogenase CrFe protein and MnFe protein were purified from a mutant strain UW3 of Azotobacter vinelandii Lipmann grown on a medium containing Cr and Mn, respectively. In order to meet the requirement for crystal growth Of O-2-susceptible proteins including nitrogenase in space, crystallization conditions were optimized for the proteins using a simple and suitable device, as a replacement for the cumbersome anaerobic box (dry box), for anaerobic addition of the protein samples. In all used precipitant and protein solutions added in the simplified plexi glass box, CrFe protein and MnFe protein could be crystallized on the spacecraft in one week by the liquid/liquid diffusion method and vapor diffusion by the sitting drop method, respectively. All formed crystals were single on the spacecraft, but under the same condition twin crystals appeared on the ground. The size of the largest crystal grown in space from CrFe protein was 2-fold larger than that on the ground. But the size of the largest crystal grown in space from MnFe protein was not larger than that on the ground. The difference in crystal growth in space between CrFe protein and MnFe protein could be resulted from the crystallization method, rather than the kind of protein.展开更多
By using the liquid/liquid diffusion method at a suitable crystallization conditions, large single and dark brown crystals (the sides of the largest crystals were 0.20 mm x 0.20 mm x 0.07 min and 0.18 mm x 0.18 mm x 0...By using the liquid/liquid diffusion method at a suitable crystallization conditions, large single and dark brown crystals (the sides of the largest crystals were 0.20 mm x 0.20 mm x 0.07 min and 0.18 mm x 0.18 mm x 0.05 mm, respectively) could be obtained from the solutions of nitrogenase CrFe protein and MnFe protein purified from a mutant UW3 of Azotobacter vinelandii Lipmarm grown in Cr- or Mn-containing but NH3-free medium. The time of crystal formation, as well as the number, size, shape and quality of crystals obviously depended on the concentrations of PEG, MgCl2 and NaCl. The liquid/liquid diffusion method seems to benefit CrFe protein and MnFe protein for the growth of large single crystals for X-ray diffraction analysis.展开更多
Under a suitable condition of crystallization, dark brown rhombohedron crystals (the lengths of the longest two diagonals were 0.25 and 0.12 mm, respectively) could be obtained from nitrogenase CrFe protein purified f...Under a suitable condition of crystallization, dark brown rhombohedron crystals (the lengths of the longest two diagonals were 0.25 and 0.12 mm, respectively) could be obtained from nitrogenase CrFe protein purified from a mutant UW3 of Azotobacter vinelandii Lipmann grown in Cr-containing but NH3-free Medium, The possibility of crystallization, as well as the. number, size and quality of crystals obviously depended on the concentrations of PEG 8000, MgCl2, NaCl, Tris and Hepes buffer, and methods of crystallization. The optimum concentrations of the chemicals for crystallization of CrFe protein were slightly different from those for crystallization of MnFe protein from UW3 grown in Mn and DeltanifZ MoFe protein from a nifZ deleted strain of A. vinelandii. The crystal seemed to be formed from CrFe protein.展开更多
The iron molybdenum cofactor(FeMoco) of nitrogenase MoFe protein from Azotobacter vinelandii OP was extracted by N methylformamide(NMF). The effects of FeMoco(in NMF) on electronic spectrum and fluorescence...The iron molybdenum cofactor(FeMoco) of nitrogenase MoFe protein from Azotobacter vinelandii OP was extracted by N methylformamide(NMF). The effects of FeMoco(in NMF) on electronic spectrum and fluorescence intensity of fluorescein dimercury acetate(FDMA)(in 1 mol/L NaOH) were investigated by fluorophotometric titrations and compared with those of (NH 4) 2MoS 4 and complexes of (NH 4) 2MoS 4 with Na 2S or Na 2S 2 or (NH 4) 2S x on the relative properties of FDMA. It was found that the electronic spectrum of FDMA displayed hypsochromic shift(17 nm) in the presence of FeMoco just like that in the presence of other inorganic sulfides and that the titration curve for the quench of FDMA with FeMoco is very similar to that for the quench of FDMA with complex of (NH 4) 2MoS 4 with Na 2S 2(mole ratio is 1∶3). The results showed that FeMoco(N) probably contained S—S bonds and its structure was found to be changed compared with Kim Rees structural model. This change should profit the formation of the polymer.展开更多
Compound(Ph_4P)[V_3MP_6](MPH_2=o-HOC_6H_4SH)was obtained by reaction of VCl_3 and Na_2MP in ethanol in the presence of Ph_4PBr.It is triclinic and crystallizes in space group P1,fw=1237.3,a=14.127(4), b=14.342(4),c=15...Compound(Ph_4P)[V_3MP_6](MPH_2=o-HOC_6H_4SH)was obtained by reaction of VCl_3 and Na_2MP in ethanol in the presence of Ph_4PBr.It is triclinic and crystallizes in space group P1,fw=1237.3,a=14.127(4), b=14.342(4),c=15.878(4);α=65.08(2),β=73.09(2),T=78.68(2)°;V=2781.3~3, Z=2,d_c=1.48 g/cm^3.Final R factor is 0.063.The three vanadium atoms are linearly arranged and bridged by the oxygen atoms and terminally chelated by the thiolato-atoms of the six MP^2-ligands in pseudo-S_6 symmetry.展开更多
The function of biomacromolecule is dependent on its space structure.X-ray diffraction analysis is generally an important way to obtain structural information of biomacromolecules.Here,the main advances in the growth ...The function of biomacromolecule is dependent on its space structure.X-ray diffraction analysis is generally an important way to obtain structural information of biomacromolecules.Here,the main advances in the growth and X-ray diffraction analysis of nitrogenase crystals are briefly introduced and reviewed.At last,the challenge and prospect of nitrogenase crystallography are discussed.展开更多
Peanuts are important oilseed legume crops that are susceptible to contamination by Aspergillus flavus in soil,leading to serious economic losses.Previously,our research team developed the Aspergillus-Rihizobia coupli...Peanuts are important oilseed legume crops that are susceptible to contamination by Aspergillus flavus in soil,leading to serious economic losses.Previously,our research team developed the Aspergillus-Rihizobia coupling(ARC)microbial inoculants and found it can reduce A.flavus abundance in the soil and promote efficient nodulation in peanuts.However,the impact of ARC microbial inoculants on different resistant varieties of A.flavus remains unclear.In this study,we screened peanut varieties that were resistant and susceptible to A.flavus and evaluated their nodulation ability and growth performance after ARC microbial inoculants treatment in the field.The results demonstrated that the nodule number and nitrogenase activity of both varieties significantly increased after ARC microbial inoculants treatment,with the highly susceptible variety AH24 showing a greater increase.For photosynthetic parameters,both varieties also increased after ARC microbial inoculants treatment,but the increase was greater in the moderately resistant variety AH1 than in the highly susceptible variety AH24.Finally,we found that the yield and yield-related traits of the moderately resistant variety AH1 were better than those of the highly susceptible variety AH24.After ARC microbial inoculants treatment,the yield traits of both peanut varieties still increased significantly,but the degree of increase of the moderately resistant variety AH1 was smaller than that of the highly susceptible variety AH24.In addition,the abundance of A.flavus in the rhizosphere soil of the two varieties significantly decreased after ARC microbial inoculants treatment,with no significant difference between the varieties.These results indicated that ARC microbial inoculants exert differential effects on the nodulation and growth of different resistant peanut varieties and have a better effect on highly susceptible varieties.These results provide a solid theoretical basis for the efficient use of ARC microbial inoculants in the field of peanuts in the future.展开更多
Nitrogenase is a metalloprotein complex that catalyses the reaction of biological nitrogen fixation. At least three genetically distinct nitrogenase systems have been confirmed in diazotrophs, namely Nil, Vnf, and Anf...Nitrogenase is a metalloprotein complex that catalyses the reaction of biological nitrogen fixation. At least three genetically distinct nitrogenase systems have been confirmed in diazotrophs, namely Nil, Vnf, and Anf, in which the active-site central metals are Mo, V, and Fe, respectively. The present review summarizes progress on the genetic, structural, and functional investigations into the three nitrogenases and discusses the possibility of the existence of other novel nitrogenases.展开更多
To identify the unknown proteins that would contaminate the α- and β-subunits of nitrogenase MoFe protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE）, the partially purified MoFe prote...To identify the unknown proteins that would contaminate the α- and β-subunits of nitrogenase MoFe protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE）, the partially purified MoFe protein （Avl） preparation was obtained from Azotobacter vinelandii Lipmann OP by chroma- tography on DEAE-cellulose （DE52） and Sephacryl S-200 columns and analyzed by PAGE and matrix- assisted laser desorption/ionization time-of-flight （MALDI-TOF） mass spectrometry. The Av 1 preparation was shown to have two main bands at the position of the α- and β-subunits of crystalline Avl on the SDS gel. However, on the anoxic native PAGE, in addition to the Avl band, the preparation was shown to have three other main bands that migrated slower than Av 1. Of these three main bands, the protein with the fastest migration was identified as bacterioferritin elsewhere. The proteins on the other two bands, termed Upper and Middle, were suggested to be two different homopolymers with the same apparent subunit electrophoretic mobilities as the α- and β-subunits of Avl, respectively. By analysis of MALDI-TOF mass spectrometry, the Upper was identified as GroEL, which belongs to the heat shock protein 60 family, and the Middle was identified as glucose-6-phosphate isomerase （PGI）. In our preparation, anoxic native electrophoresis indicated that GroEL was composed of 14 identical subunits and that PGI was composed of 10 identical subunits. This is the first report of PGI, with so many subunits. The contaminating proteins in the Av 1 preparation, mainly GroEL and PGI, could be totally or partially removed from Av 1 if the shoulders and center of the elution peak were collected separately from the Sephacryl S-200 column and the center fraction was purified further by Q-Sepharose developed with an NaC1 concentration gradient. Thus, Avl with more than 90% purity was obtained. Obviously, this modified method is useful for the purification of mutant MoFe proteins with a high purity.展开更多
The problem of how the nitrogenase is capable of transforming dinitrogen into ammoniahas indeed become one of the hot subjects in the field of molecular biochemistry. In1992, Kim and Rees of the California Institute o...The problem of how the nitrogenase is capable of transforming dinitrogen into ammoniahas indeed become one of the hot subjects in the field of molecular biochemistry. In1992, Kim and Rees of the California Institute of Technology published their result of展开更多
RECENTLY,the structure of MoFe protein from Azotobacter vinelandii has been deter-mined,and based on this,the structural models of FeMo-co and P-clusters in MoFe proteinhave been proposed.According to the model,FeMo-c...RECENTLY,the structure of MoFe protein from Azotobacter vinelandii has been deter-mined,and based on this,the structural models of FeMo-co and P-clusters in MoFe proteinhave been proposed.According to the model,FeMo-co consists of two clusters,Fe<sub>4</sub>S<sub>3</sub> andMoFe<sub>3</sub>S<sub>3</sub> with a homocitrate ligand coordinated to Mo atom,bridged by three nonprotein lig-ands,two of which are inorganic sulfur atoms and the other is nitrogen- or展开更多
Dear Editor,Biological soil crusts(BSCs),a layered structure formed by associations of soil organisms and topsoil,dominate arid and semiarid areas and serve important ecological functions in these areas(Eldridge and G...Dear Editor,Biological soil crusts(BSCs),a layered structure formed by associations of soil organisms and topsoil,dominate arid and semiarid areas and serve important ecological functions in these areas(Eldridge and Greene,1994).Nitrogen fixation by BSCs is the main source of N in arid and semi-arid ecosystems.Desiccation is the most notable factor that influences BSCs,which recover physiological activity only after moistening.By influencing the amount of carbohydrates,展开更多
Complex(n-Bu_4N)[Mo_2O_2(OMe)(mp)_3][1,mp=(o-OC_(?)H_4S)^(2-)]was synthesized by the reaction of MoOCl_3(THF)_2 and Na_2mp in EtOH and crystallized in monoclinic space group P2_(?)/n with crystal data:a=13.910(3),b=23...Complex(n-Bu_4N)[Mo_2O_2(OMe)(mp)_3][1,mp=(o-OC_(?)H_4S)^(2-)]was synthesized by the reaction of MoOCl_3(THF)_2 and Na_2mp in EtOH and crystallized in monoclinic space group P2_(?)/n with crystal data:a=13.910(3),b=23.554(3),c=12.558(2)(?),β=105.20(2)°,Z=4,D_o=1.455g/ cm^3,final R=0.077 for 5325 reflections[I>3σ(I)].The two[Mo(O)mp]^+ moicties are bridged by one 1,2-bidentate ligand mp^(2-) with exceedingly small bite angle(69.0°)and bite distance (2.70(?))and by a methoxy group.The structure can be interpreted as two distorted octahedra around the Mo atoms sharing a face.Attempts have been made to provide structural informa- tions for the syntheses of modelling compounds of FeMoco by comparing the Mo—O and Mo—S bond distances of o-mercaptophenolate ligated compounds to those of the molybdenum site of nitrogenase.展开更多
Effects of water stress and foliar boron (FB) application on soybean (Glycine max (L) Merr.) seed composition and nitrogen metabolism have not been well investigated. Therefore, the objective of this study was to inve...Effects of water stress and foliar boron (FB) application on soybean (Glycine max (L) Merr.) seed composition and nitrogen metabolism have not been well investigated. Therefore, the objective of this study was to investigate the effects of water stress and FB on seed protein, oil, fatty acids, nitrate reductase activity (NRA), and nitrogenase activity (NA). A repeated greenhouse experiment was conducted where one set of soybean plants were subjected to water stress (WS), and the other set was watered (W). Foliar boron (B) was applied at rate of 0.45 kg·ha-1. Treatments were watered-plants with no FB (W), watered-plants with FB (WB), water-stress plants with no FB (WS), and water-stress plants with FB (WSB). The results showed that seed protein and oil percentage were significantly (P 15N/ 14N and 13C/12C natural abundance were altered between watered-and watered-stressed plants. These results suggest that water stress and FB can influence seed composition, and nitrogen metabolism, and 15N/14N and 13C/12C ratios, reflecting environmental and metabolic changes in carbon and nitrogen fixation pathways. Lack of B translocation from leaves to seed under water stress may suggest a possible mechanism of limited B translocation under water stress. These findings may be beneficial to breeders to select for B translocation efficiency under drought conditions. Altered 15N/14N and 13C/12C under water stress can be used as a tool to select for drought tolerance using N and C isotopes in the breeding programs.展开更多
The microbial agent ARC-BBBE demonstration trials were conducted in four provinces in the main peanutproducing areas of the Huang-huai-hai plain of China.The results revealed that the application of ARC-BBBE led to a ...The microbial agent ARC-BBBE demonstration trials were conducted in four provinces in the main peanutproducing areas of the Huang-huai-hai plain of China.The results revealed that the application of ARC-BBBE led to a 1.09–1.70 fold increase in the number of nodules in the treatment group at the demonstration site compared to the control group.Moreover,the nodule weight in the treatment group was 0.80–3.32 times higher than that of the control group,and nitrogenase activity per plant showed a significant enhancement by 1.00–2.83 fold compared to controls.Additionally,notable improvements were observed in terms of increased fresh weight of whole plants,well-filled pod numbers,and enhanced growth performance;ultimately resulting in a harvest yield increase ranging from 9.46%to 49.04%.The abundance of Aspergillus flavus in rhizosphere soil was determined by the dilution spread plate method,and the inhibition rate was up to 86.7%.The application of ARC-BBBE in the significant peanut-producing areas of Huang-huai-hai has effects of promoting growth,nodulation,and increasing production.At the same time,it has the effect of inhibiting and controlling soil Aspergillus flavus,which provides a new green and low-carbon way to promote the high-quality development of the peanut industry.展开更多
Reactions of potassium molybdate with racemic malic acid （H3mal = C4H6O5） result in the isolation of two mesomeric molybdenum malate complexes K8[（MoO2）2O（R-mal）2][（MoO2）2O（Smal）2]-4H2O 1 and （Him）2K6[（M...Reactions of potassium molybdate with racemic malic acid （H3mal = C4H6O5） result in the isolation of two mesomeric molybdenum malate complexes K8[（MoO2）2O（R-mal）2][（MoO2）2O（Smal）2]-4H2O 1 and （Him）2K6[（MoO2）4O3（R-mal）2][（MoOE）4O3（S-mal）2]-8H2O 2. Complex 1 belongs to the monoclinic system, space group C2/c with a = 14.8637（3）, b = 6.9544（1）, c = 19.6783（5）A, β = 100.081（2）°, V = 2002.70（7） A^3, Mr = 1452.88, Z = 2, F（000） = 1416, T = 173 K, Dc = 2.409 g/cm3, fl（MoKa＇） = 2.167, R = 0.0283 and wR = 0.0733.2 is of triclinic system, space group P1^- with a = 8.7707（2）, b = 9.3310（3）, c = 17.9093（7）A, α= 83.781（3）, β = 85.626（2）, y= 84.822（2）°, V = 1447.84（8）A^3, Mr = 2160.68, Z = 1, F（000） = 1048, T = 173 K, Dc = 2.478 g/cm^3,μ（MoKα） = 2.230, R = 0.0234 and wR = 0.0584.1 is the first isolated dinuclear molybdenum（VI） malato complex in 1：1 molar ratio. The molybdenum atoms in the two complexes are six-coordinated in an approximately octahedral geometry. Two malates coordinate tridentately with the Mo atom via their α-alkoxy, α-carboxy and α-carboxy groups in 1 and 2. β-Carboxy group in 2 further links with the other two Mo atoms to give a tetrameric unit. The solution ^1H and ^13C NMR spectra indicate that dimeric malate molybdenum in 1 dissociates partly in solution and exists in an equilibrium with tetrameric species, while 2 is stable and retains its tetrameric structure without any dissociation.展开更多
文摘Under a suitable condition of crystallization, dark brown short rhombohedron crystals could be obtained from FeMoco-deficient MoFe protein (DeltanifE Avl) purified from a nifE deleted mutant DJ35 of Azotobacter vinelandii Lipmann grown in NH3-limited medium. The number, size and quality of crystals were significantly affected by either the concentration of precipitants and buffer or diffusion method. The longest sides of the largest crystal of DeltanifE Avl protein, which was obtained by vapor diffusion in the hanging drop method, were 0.12 and 0.13 mm, respectively.
文摘Under a suitable condition of crystallization, dark brown short rhombohedron crystals could be obtained from nitrogenase MnFe protein purified from a mutant UW3 of Azotobacter vinelandii Lipmann grown in Mn-containing but Mo- and NH3-free medium. The possibility of crystallization, and number, size and quality of crystals were obviously dependent on concentrations of NaCl, MgCl2, PEG 8000,Tris and Hepes buffer and on methods for crystallization. PEG concentration affected on the shape of the crystals. The optimal, concentrations of the chemicals for crystallization of MnFe protein were slightly different from those for crystallization of Delta nifZ MoFe protein from a nifZ deleted strain of Azotobacter vinelandii. SDS-PAGE showed that the protein from the dissolved crystals was almost the same as MnFe protein before crystallization, indicating that the crystal was formed from MnFe protein.
文摘A mutant UW 3, which is unable to fix N 2 in the presence of Mo (Nif -) but undergo phenotypic reversal to Nif + under Mo deficiency, was able to grow in Mo- and NH 3-deficient medium containing Mn, and the growth was accelerated by Mn at low concentration. A partly purified nitrogenase component Ⅰ protein separated from UW 3 grown in the Mn-containing medium was shown to contain Fe and Mn atoms (ratio of Fe/Mo/Mn: 10.41/0.19/1.00) with C 2H 2- and H +-reducing activity which almost equal to half of that of MoFe protein purified from wild-type mutant of Azotobacter vinelandii Lipmann. This protein was obviously different from MoFe protein in both absorption spectrum and circular dichroism, and the molecular weight of subunits in Mn-containing protein was close to that of α subunit in MoFe protein. The preliminary results indicated that the protein containing Mn might be a nitrogenase component Ⅰ protein.
文摘A mutant UW 3, which is unable to fix N 2 in the presence of Mo (Nif -) but can undergo phenotypic reversal to Nif + under Mo_deficient conditions, was able to grow in Cr_containing but Mo_ and NH 3_deficient medium. A partly purified nitrogenase component Ⅰ protein obtained from UW 3 grown on the Cr_containing medium was shown to contain Fe and Cr (atom ratio of Fe to Cr and Mo to Cr: 11.60 and 0.41) and to have 70% of the C 2H 2_ and H +_reduction activity of MoFe protein from the wild_type strain of Azotobacter vinelandii Lipmann. The Cr_containing protein was different in subunit composition from that of MnFe protein purified from the mutant strain grown in the presence of Mn, but similar to that of MoFe protein, that is, it was a tetramer composed of two different subunits (α 2β 2). The preliminary results indicated that the Cr_containing protein might be a nitrogenase component Ⅰ protein.
文摘Nitrogenase CrFe protein and MnFe protein were purified from a mutant strain UW3 of Azotobacter vinelandii Lipmann grown on a medium containing Cr and Mn, respectively. In order to meet the requirement for crystal growth Of O-2-susceptible proteins including nitrogenase in space, crystallization conditions were optimized for the proteins using a simple and suitable device, as a replacement for the cumbersome anaerobic box (dry box), for anaerobic addition of the protein samples. In all used precipitant and protein solutions added in the simplified plexi glass box, CrFe protein and MnFe protein could be crystallized on the spacecraft in one week by the liquid/liquid diffusion method and vapor diffusion by the sitting drop method, respectively. All formed crystals were single on the spacecraft, but under the same condition twin crystals appeared on the ground. The size of the largest crystal grown in space from CrFe protein was 2-fold larger than that on the ground. But the size of the largest crystal grown in space from MnFe protein was not larger than that on the ground. The difference in crystal growth in space between CrFe protein and MnFe protein could be resulted from the crystallization method, rather than the kind of protein.
文摘By using the liquid/liquid diffusion method at a suitable crystallization conditions, large single and dark brown crystals (the sides of the largest crystals were 0.20 mm x 0.20 mm x 0.07 min and 0.18 mm x 0.18 mm x 0.05 mm, respectively) could be obtained from the solutions of nitrogenase CrFe protein and MnFe protein purified from a mutant UW3 of Azotobacter vinelandii Lipmarm grown in Cr- or Mn-containing but NH3-free medium. The time of crystal formation, as well as the number, size, shape and quality of crystals obviously depended on the concentrations of PEG, MgCl2 and NaCl. The liquid/liquid diffusion method seems to benefit CrFe protein and MnFe protein for the growth of large single crystals for X-ray diffraction analysis.
文摘Under a suitable condition of crystallization, dark brown rhombohedron crystals (the lengths of the longest two diagonals were 0.25 and 0.12 mm, respectively) could be obtained from nitrogenase CrFe protein purified from a mutant UW3 of Azotobacter vinelandii Lipmann grown in Cr-containing but NH3-free Medium, The possibility of crystallization, as well as the. number, size and quality of crystals obviously depended on the concentrations of PEG 8000, MgCl2, NaCl, Tris and Hepes buffer, and methods of crystallization. The optimum concentrations of the chemicals for crystallization of CrFe protein were slightly different from those for crystallization of MnFe protein from UW3 grown in Mn and DeltanifZ MoFe protein from a nifZ deleted strain of A. vinelandii. The crystal seemed to be formed from CrFe protein.
文摘The iron molybdenum cofactor(FeMoco) of nitrogenase MoFe protein from Azotobacter vinelandii OP was extracted by N methylformamide(NMF). The effects of FeMoco(in NMF) on electronic spectrum and fluorescence intensity of fluorescein dimercury acetate(FDMA)(in 1 mol/L NaOH) were investigated by fluorophotometric titrations and compared with those of (NH 4) 2MoS 4 and complexes of (NH 4) 2MoS 4 with Na 2S or Na 2S 2 or (NH 4) 2S x on the relative properties of FDMA. It was found that the electronic spectrum of FDMA displayed hypsochromic shift(17 nm) in the presence of FeMoco just like that in the presence of other inorganic sulfides and that the titration curve for the quench of FDMA with FeMoco is very similar to that for the quench of FDMA with complex of (NH 4) 2MoS 4 with Na 2S 2(mole ratio is 1∶3). The results showed that FeMoco(N) probably contained S—S bonds and its structure was found to be changed compared with Kim Rees structural model. This change should profit the formation of the polymer.
文摘Compound(Ph_4P)[V_3MP_6](MPH_2=o-HOC_6H_4SH)was obtained by reaction of VCl_3 and Na_2MP in ethanol in the presence of Ph_4PBr.It is triclinic and crystallizes in space group P1,fw=1237.3,a=14.127(4), b=14.342(4),c=15.878(4);α=65.08(2),β=73.09(2),T=78.68(2)°;V=2781.3~3, Z=2,d_c=1.48 g/cm^3.Final R factor is 0.063.The three vanadium atoms are linearly arranged and bridged by the oxygen atoms and terminally chelated by the thiolato-atoms of the six MP^2-ligands in pseudo-S_6 symmetry.
文摘The function of biomacromolecule is dependent on its space structure.X-ray diffraction analysis is generally an important way to obtain structural information of biomacromolecules.Here,the main advances in the growth and X-ray diffraction analysis of nitrogenase crystals are briefly introduced and reviewed.At last,the challenge and prospect of nitrogenase crystallography are discussed.
基金supported by the Agricultural Science and Technology Innovation Program(CAAS-ZDRW202416)the Foundation of Hubei Hongshan Laboratory(2021hszd015)+1 种基金the Science and Technology Major Projects of Hubei Province(2023BBA002)the Knowledge Innovation Program of Wuhan-Basi Research(2023020201010126)。
文摘Peanuts are important oilseed legume crops that are susceptible to contamination by Aspergillus flavus in soil,leading to serious economic losses.Previously,our research team developed the Aspergillus-Rihizobia coupling(ARC)microbial inoculants and found it can reduce A.flavus abundance in the soil and promote efficient nodulation in peanuts.However,the impact of ARC microbial inoculants on different resistant varieties of A.flavus remains unclear.In this study,we screened peanut varieties that were resistant and susceptible to A.flavus and evaluated their nodulation ability and growth performance after ARC microbial inoculants treatment in the field.The results demonstrated that the nodule number and nitrogenase activity of both varieties significantly increased after ARC microbial inoculants treatment,with the highly susceptible variety AH24 showing a greater increase.For photosynthetic parameters,both varieties also increased after ARC microbial inoculants treatment,but the increase was greater in the moderately resistant variety AH1 than in the highly susceptible variety AH24.Finally,we found that the yield and yield-related traits of the moderately resistant variety AH1 were better than those of the highly susceptible variety AH24.After ARC microbial inoculants treatment,the yield traits of both peanut varieties still increased significantly,but the degree of increase of the moderately resistant variety AH1 was smaller than that of the highly susceptible variety AH24.In addition,the abundance of A.flavus in the rhizosphere soil of the two varieties significantly decreased after ARC microbial inoculants treatment,with no significant difference between the varieties.These results indicated that ARC microbial inoculants exert differential effects on the nodulation and growth of different resistant peanut varieties and have a better effect on highly susceptible varieties.These results provide a solid theoretical basis for the efficient use of ARC microbial inoculants in the field of peanuts in the future.
基金Supported by the State Key Basic Research and DevePopment Plan of China （001CB1089-06） and the NationaP NaturaP Science Foundation of China （30270296）.
文摘Nitrogenase is a metalloprotein complex that catalyses the reaction of biological nitrogen fixation. At least three genetically distinct nitrogenase systems have been confirmed in diazotrophs, namely Nil, Vnf, and Anf, in which the active-site central metals are Mo, V, and Fe, respectively. The present review summarizes progress on the genetic, structural, and functional investigations into the three nitrogenases and discusses the possibility of the existence of other novel nitrogenases.
文摘To identify the unknown proteins that would contaminate the α- and β-subunits of nitrogenase MoFe protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE）, the partially purified MoFe protein （Avl） preparation was obtained from Azotobacter vinelandii Lipmann OP by chroma- tography on DEAE-cellulose （DE52） and Sephacryl S-200 columns and analyzed by PAGE and matrix- assisted laser desorption/ionization time-of-flight （MALDI-TOF） mass spectrometry. The Av 1 preparation was shown to have two main bands at the position of the α- and β-subunits of crystalline Avl on the SDS gel. However, on the anoxic native PAGE, in addition to the Avl band, the preparation was shown to have three other main bands that migrated slower than Av 1. Of these three main bands, the protein with the fastest migration was identified as bacterioferritin elsewhere. The proteins on the other two bands, termed Upper and Middle, were suggested to be two different homopolymers with the same apparent subunit electrophoretic mobilities as the α- and β-subunits of Avl, respectively. By analysis of MALDI-TOF mass spectrometry, the Upper was identified as GroEL, which belongs to the heat shock protein 60 family, and the Middle was identified as glucose-6-phosphate isomerase （PGI）. In our preparation, anoxic native electrophoresis indicated that GroEL was composed of 14 identical subunits and that PGI was composed of 10 identical subunits. This is the first report of PGI, with so many subunits. The contaminating proteins in the Av 1 preparation, mainly GroEL and PGI, could be totally or partially removed from Av 1 if the shoulders and center of the elution peak were collected separately from the Sephacryl S-200 column and the center fraction was purified further by Q-Sepharose developed with an NaC1 concentration gradient. Thus, Avl with more than 90% purity was obtained. Obviously, this modified method is useful for the purification of mutant MoFe proteins with a high purity.
基金Project supported by the National Natural Science Foundation of China as well as the State Key Laboratory Foundation of Structural Chemistry.
文摘The problem of how the nitrogenase is capable of transforming dinitrogen into ammoniahas indeed become one of the hot subjects in the field of molecular biochemistry. In1992, Kim and Rees of the California Institute of Technology published their result of
文摘RECENTLY,the structure of MoFe protein from Azotobacter vinelandii has been deter-mined,and based on this,the structural models of FeMo-co and P-clusters in MoFe proteinhave been proposed.According to the model,FeMo-co consists of two clusters,Fe<sub>4</sub>S<sub>3</sub> andMoFe<sub>3</sub>S<sub>3</sub> with a homocitrate ligand coordinated to Mo atom,bridged by three nonprotein lig-ands,two of which are inorganic sulfur atoms and the other is nitrogen- or
基金supported by the Special Fund for Forest Scientific Research in the Public Welfare of China (201404204-02)
文摘Dear Editor,Biological soil crusts(BSCs),a layered structure formed by associations of soil organisms and topsoil,dominate arid and semiarid areas and serve important ecological functions in these areas(Eldridge and Greene,1994).Nitrogen fixation by BSCs is the main source of N in arid and semi-arid ecosystems.Desiccation is the most notable factor that influences BSCs,which recover physiological activity only after moistening.By influencing the amount of carbohydrates,
基金Project supported by the National Natural Science Foundation of Chinathe Natural Science Foundation of the Chinese Academy of Sciences the Natural Science Foundation of Fujian Province.
文摘Complex(n-Bu_4N)[Mo_2O_2(OMe)(mp)_3][1,mp=(o-OC_(?)H_4S)^(2-)]was synthesized by the reaction of MoOCl_3(THF)_2 and Na_2mp in EtOH and crystallized in monoclinic space group P2_(?)/n with crystal data:a=13.910(3),b=23.554(3),c=12.558(2)(?),β=105.20(2)°,Z=4,D_o=1.455g/ cm^3,final R=0.077 for 5325 reflections[I>3σ(I)].The two[Mo(O)mp]^+ moicties are bridged by one 1,2-bidentate ligand mp^(2-) with exceedingly small bite angle(69.0°)and bite distance (2.70(?))and by a methoxy group.The structure can be interpreted as two distorted octahedra around the Mo atoms sharing a face.Attempts have been made to provide structural informa- tions for the syntheses of modelling compounds of FeMoco by comparing the Mo—O and Mo—S bond distances of o-mercaptophenolate ligated compounds to those of the molybdenum site of nitrogenase.
文摘Effects of water stress and foliar boron (FB) application on soybean (Glycine max (L) Merr.) seed composition and nitrogen metabolism have not been well investigated. Therefore, the objective of this study was to investigate the effects of water stress and FB on seed protein, oil, fatty acids, nitrate reductase activity (NRA), and nitrogenase activity (NA). A repeated greenhouse experiment was conducted where one set of soybean plants were subjected to water stress (WS), and the other set was watered (W). Foliar boron (B) was applied at rate of 0.45 kg·ha-1. Treatments were watered-plants with no FB (W), watered-plants with FB (WB), water-stress plants with no FB (WS), and water-stress plants with FB (WSB). The results showed that seed protein and oil percentage were significantly (P 15N/ 14N and 13C/12C natural abundance were altered between watered-and watered-stressed plants. These results suggest that water stress and FB can influence seed composition, and nitrogen metabolism, and 15N/14N and 13C/12C ratios, reflecting environmental and metabolic changes in carbon and nitrogen fixation pathways. Lack of B translocation from leaves to seed under water stress may suggest a possible mechanism of limited B translocation under water stress. These findings may be beneficial to breeders to select for B translocation efficiency under drought conditions. Altered 15N/14N and 13C/12C under water stress can be used as a tool to select for drought tolerance using N and C isotopes in the breeding programs.
基金The authors thank the National Natural Sciences Foundation of China(32030085,U22A20551)Major project of Hubei Hongshan Laboratory(2021hszd015)+1 种基金Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences(CAAS-ASTIP2021-OCRI)Knowledge Innovation Program of Wuhan-Shuguang Project(3570).
文摘The microbial agent ARC-BBBE demonstration trials were conducted in four provinces in the main peanutproducing areas of the Huang-huai-hai plain of China.The results revealed that the application of ARC-BBBE led to a 1.09–1.70 fold increase in the number of nodules in the treatment group at the demonstration site compared to the control group.Moreover,the nodule weight in the treatment group was 0.80–3.32 times higher than that of the control group,and nitrogenase activity per plant showed a significant enhancement by 1.00–2.83 fold compared to controls.Additionally,notable improvements were observed in terms of increased fresh weight of whole plants,well-filled pod numbers,and enhanced growth performance;ultimately resulting in a harvest yield increase ranging from 9.46%to 49.04%.The abundance of Aspergillus flavus in rhizosphere soil was determined by the dilution spread plate method,and the inhibition rate was up to 86.7%.The application of ARC-BBBE in the significant peanut-producing areas of Huang-huai-hai has effects of promoting growth,nodulation,and increasing production.At the same time,it has the effect of inhibiting and controlling soil Aspergillus flavus,which provides a new green and low-carbon way to promote the high-quality development of the peanut industry.
基金the Ministry of Science & Technology (2005CB221408)National Natural Science Foundation of China (20423002, 205710617) for financial support.
文摘Reactions of potassium molybdate with racemic malic acid （H3mal = C4H6O5） result in the isolation of two mesomeric molybdenum malate complexes K8[（MoO2）2O（R-mal）2][（MoO2）2O（Smal）2]-4H2O 1 and （Him）2K6[（MoO2）4O3（R-mal）2][（MoOE）4O3（S-mal）2]-8H2O 2. Complex 1 belongs to the monoclinic system, space group C2/c with a = 14.8637（3）, b = 6.9544（1）, c = 19.6783（5）A, β = 100.081（2）°, V = 2002.70（7） A^3, Mr = 1452.88, Z = 2, F（000） = 1416, T = 173 K, Dc = 2.409 g/cm3, fl（MoKa＇） = 2.167, R = 0.0283 and wR = 0.0733.2 is of triclinic system, space group P1^- with a = 8.7707（2）, b = 9.3310（3）, c = 17.9093（7）A, α= 83.781（3）, β = 85.626（2）, y= 84.822（2）°, V = 1447.84（8）A^3, Mr = 2160.68, Z = 1, F（000） = 1048, T = 173 K, Dc = 2.478 g/cm^3,μ（MoKα） = 2.230, R = 0.0234 and wR = 0.0584.1 is the first isolated dinuclear molybdenum（VI） malato complex in 1：1 molar ratio. The molybdenum atoms in the two complexes are six-coordinated in an approximately octahedral geometry. Two malates coordinate tridentately with the Mo atom via their α-alkoxy, α-carboxy and α-carboxy groups in 1 and 2. β-Carboxy group in 2 further links with the other two Mo atoms to give a tetrameric unit. The solution ^1H and ^13C NMR spectra indicate that dimeric malate molybdenum in 1 dissociates partly in solution and exists in an equilibrium with tetrameric species, while 2 is stable and retains its tetrameric structure without any dissociation.
