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1.
生物大分子的功能取决于它的空间结构.X射线衍射分析是获得生物大分子结构信息的常用方法.本文对固氮酶晶体的生长及其X射线衍射分析的主要进展简要地进行了介绍和评论.最后展望了今后的发展及问题. 相似文献
2.
生物大分子的功能取决于它的空间结构。X射线衍射分析是获得生物大分子结构信息的常用方法。本文对固氮酶晶体的生长及其X射线衍射分析的主要进展简要地进行了介绍和评论。最后展望了今后的发展及问题。 相似文献
3.
棕色固氮菌(Azotobacter vinelandii Lipmann)突变种UW3能在无Mo的无氨培养基中固氮生长,低浓度的Mn对UW3突变种生长有促进作用,从在Mn中生长的UW3菌体中分离得到的部分纯固氮酶组分Ⅰ蛋白含量有Mn和Fe原子(Fe/Mo/Mn为10.41:0.19:1.00)并有OP MoFe蛋白一半的还原乙炔和质子的活性。这种蛋白的吸收光谱和圆二色谱与MoFe蛋白存在明显的差异,含Mn蛋白的亚基分子量都与MoFe蛋白的α亚基相近。初步结果表明,这种含Mn蛋白可胡是一种固氮酶组分Ⅰ蛋白。 相似文献
4.
固氮酶CrFe蛋白和MnFe蛋白的空间晶体生长 总被引:3,自引:0,他引:3
从分别牛长于含Mn和Cr培养基中的棕色固氮菌(Azotobacter vinelandii Lipmann)突变种UW3分离纯化出MnFe和CrFe蛋白。为适应包括固氮酶在内的氧敏感蛋白的空间晶体生长的要求,应用简易而适用的厌氧加样装置代替固氮酶实验室所用的笨重厌氧箱(dry box),在地面进行厌氧加样。在充满氮气的简便有机玻璃箱内厌氧加样的所有样品中,分别用液/液扩散法和汽相扩散的坐滴法都可在一周内使MnFe和CrFe蛋白在宇宙飞船上从溶液中结晶出来。在所用的数种蛋白沉淀剂中,飞船上形成的所有晶体都为单品,而地面上在多数沉淀剂中都生成大量挛晶。在相同沉淀剂中用液/液扩散法,飞船上生成CrFe蛋白的最大晶体比地面生成的最大晶体大1倍。而在相同沉淀剂中用汽相扩散的坐滴法,飞船上生成的MnFe蛋白最大晶体却没有地面生成的最大晶体大。这种差异也许是由不同结晶方法而不是不同蛋白所引起的。 相似文献
5.
固氮酶固氮机理的新观点张纯喜(中国科学院福建物质结构研究所,福州350002)本文提出了一个关于固氮酶固氮机理的新观点,认为在固氮酶固氮过程中,还原态的P一簇产生活泼的氢原子,活泼的氢原子部分用于产生氢气。部分用于激活FeMo-辅基(使氧化态的FeM... 相似文献
6.
在含Mo固氮培养基中不能生长而在无Mo条件下可固氮生长的固氮菌(Azotobacter vinelandii Lipmann)突变种UW3, 能在无Mo而含Cr的无氮培养基中生长.从菌体中分离得到的部分纯固氮酶组分Ⅰ蛋白含有Cr和Fe 原子(Fe/Mo/Cr为11.60∶0.41∶1.00),并能表达相当于棕色固氮菌野生型固氮菌MoFe蛋白对乙炔和质子还原的70%的活性.与从Mn中生长的UW3菌体中所提取纯化的MnFe蛋白不同,这种含铬蛋白与MoFe蛋白(α2β2)一样,是由两种亚基组成的四聚体.初步结果表明,这种含Cr蛋白可能是一种固氮酶组分Ⅰ蛋白. 相似文献
7.
在含Mo固氮培养基中不能生长而在无Mo条件下可固氮生长的固氮菌(Azotobacter vinelandii Lipmann)突变种UW3,能在无Mo而含Cr的无氮培养基中生长。从菌体中分离得到的部分纯固氮酶组分I蛋白含有Cr和Fe原子(Fe/Mo/Cr为11.60:0.41:1.00),并能表达相当于棕色固氮菌野生型固氮菌MoFe蛋白对乙炔和质子还原的70%的活性。与从Mn中生长的UW3菌体中所提取纯化的MnFe蛋白不同,这种含铬蛋白与MoFe蛋白(α2β2)一样,是由两种亚基组成的四聚体。初步结果表明,这种含Cr蛋白可能是一种固氮酶组分I蛋白。 相似文献
8.
固氮酶由两种铁硫蛋白(钼铁蛋白和铁蛋白)组成。由还原剂提供电子经铁(Fe)蛋白传递给钼铁(MoFe)蛋白,在MoFe蛋白的活性中心部位进行N_2、C_2H_2等多种底物的还原[10,11,20]。MoFe蛋白中的Mo、Fe原子和酸不稳定性硫原子(S~*)组成2个M簇(FeM-oco)、3—4个P簇(P-cluster)及1—2个S(2Fe)簇。在底物还原过程中,这些原子簇都可能参与电子的传递。铁钼辅因子(FeMoco)已被认为是络合和还原底物的重要部位。因此,要阐明MoFe蛋白的作用机理就得研究FeMoco的结构和功 相似文献
9.
固氮酶由钼铁蛋白和铁蛋白组成 ,钼铁蛋白包含FeMoco和P Cluster。为解释固氮机理 ,Kim等 ( 1 992 )、Peters等 ( 1 997)和Mayer等 ( 1 999)分别解析出分辨率为 2 8 、2 .0 和1 .6 的钼铁蛋白晶体结构。Einsle等 ( 2 0 0 2 )又获得了 1 .1 6 的钼铁蛋白结晶 ,在对其进行结构解析后发现 :FeMoco的内部有一个轻的原子与六个铁原子键连接。此轻原子不可能是硫 ,最可能是氮 ,但不排除是碳或氧的可能。已有极好的证据表明FeMoco是固氮酶的底物结合位点和还原中心 ,但底物是如何结合到FeMoc… 相似文献
10.
在含Mo固氮培养基中不能生长而在无Mo条件下可固氮生长的固氮菌 (AzotobactervinelandiiLipmann)突变种UW3 ,能在无Mo而含Cr的无氮培养基中生长。从菌体中分离得到的部分纯固氮酶组分Ⅰ蛋白含有Cr和Fe原子 (Fe/Mo/Cr为 11.6 0∶0 .41∶1.0 0 ) ,并能表达相当于棕色固氮菌野生型固氮菌MoFe蛋白对乙炔和质子还原的 70 %的活性。与从Mn中生长的UW3 菌体中所提取纯化的MnFe蛋白不同 ,这种含铬蛋白与MoFe蛋白 (α2 β2 )一样 ,是由两种亚基组成的四聚体。初步结果表明 ,这种含Cr蛋白可能是一种固氮酶组分Ⅰ蛋白。 相似文献
11.
Increasing nitrogenase catalytic efficiency for MgATP by changing serine 16 of its Fe protein to threonine: use of Mn2+ to show interaction of serine 16 with Mg2+. 总被引:1,自引:1,他引:0
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L. C. Seefeldt L. E. Mortenson 《Protein science : a publication of the Protein Society》1993,2(1):93-102
MgATP-binding and hydrolysis are an integral part of the nitrogenase catalytic mechanism. We are exploring the function of MgATP hydrolysis in this reaction by analyzing the properties of the Fe protein (FeP) component of Azotobacter vinelandii nitrogenase altered by site-directed mutagenesis. We have previously (Seefeldt, L.C., Morgan, T.V., Dean, D.R., & Mortenson, L.E., 1992, J. Biol. Chem. 267, 6680-6688) identified a region near the N-terminus of FeP that is involved in interaction with MgATP. This region of FeP is homologous to the well-known nucleotide-binding motif GXXXXGKS/T. In the present work, we examined the function of the four hydroxyl-containing amino acids immediately C-terminal to the conserved lysine 15 that is involved in interaction with the gamma-phosphate of MgATP. We have established, by altering independently Thr 17, Thr 18, and Thr 19 to alanine, that a hydroxyl-containing residue is not needed at these positions for FeP to function. In contrast, an hydroxyl-containing amino acid at position 16 was found to be critical for FeP function. When the strictly conserved Ser 16 was altered to Ala, Cys, Asp, or Gly, the FeP did not support N2 fixation when expressed in place of the wild-type FeP in A. vinelandii. Altering Ser 16 to Thr (S16T), however, resulted in the expression of an FeP that was partially active. This S16T FeP was purified to homogeneity, and its biochemical examination allowed us to assign a catalytic function to this hydroxyl group in the nitrogenase mechanism. Of particular importance was the finding that the S16T FeP had a significantly higher affinity for MgATP than the wild-type FeP, with a measured Km of 20 microM compared to the wild-type FeP Km of 220 microM. This increased kinetic affinity for MgATP was reflected in a significantly stronger binding of the S16T FeP for MgATP. In contrast, the affinity for MgADP, which binds at the same site as MgATP, was unchanged. The catalytic efficiency (kcat/Km) of S16T FeP was found to be 5.3-fold higher than for the wild-type FeP, with the S16T FeP supporting up to 10 times greater nitrogenase activity at low MgATP concentrations. This indicates a role for the hydroxyl group at position 16 in interaction with MgATP but not MgADP. The site of interaction of this residue was further defined by examining the properties of wild-type and S16T FePs in utilizing MnATP compared with MgATP.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
12.
lvaro Eseverri Gema Lpez‐Torrejn Xi Jiang Stefan Burn Luis M. Rubio Elena Caro 《Plant biotechnology journal》2020,18(9):1882-1896
The generation of nitrogen fixing crops is considered a challenge that could lead to a new agricultural ‘green’ revolution. Here, we report the use of synthetic biology tools to achieve and optimize the production of active nitrogenase Fe protein (NifH) in the chloroplasts of tobacco plants. Azotobacter vinelandii nitrogen fixation genes, nifH, M, U and S, were re‐designed for protein accumulation in tobacco cells. Targeting to the chloroplast was optimized by screening and identifying minimal length transit peptides performing properly for each specific Nif protein. Putative peptidyl‐prolyl cis‐trans isomerase NifM proved necessary for NifH solubility in the stroma. Purified NifU, a protein involved in the biogenesis of NifH [4Fe‐4S] cluster, was found functional in NifH reconstitution assays. Importantly, NifH purified from tobacco chloroplasts was active in the reduction of acetylene to ethylene, with the requirement of nifU and nifS co‐expression. These results support the suitability of chloroplasts to host functional nitrogenase proteins, paving the way for future studies in the engineering of nitrogen fixation in higher plant plastids and describing an optimization pipeline that could also be used in other organisms and in the engineering of new metabolic pathways in plastids. 相似文献
13.
The nitrogenase Fe protein is a key component of the biochemical machinery responsible for the process of biological nitrogen fixation. The Fe protein is a member of a class of nucleotide-binding proteins that couple the binding and hydrolysis of nucleoside triphosphates to conformational changes. The nucleotide-dependent conformational changes modulate the formation of a macromolecular complex, and some members of the class include Galpha, EF-Tu, and myosin. The members of this class are highly interesting model systems for the analysis of aspects of thermal adaptability, since their mechanisms involve protein conformational change and protein-protein interactions. In this study, we have used our extensive knowledge of the structure of the Azotobacter vinelandii nitrogenase Fe protein in multiple structural conformations, and standard homology modeling approaches have been used to generate reliable models of the Fe protein from thermophilic Methanobacter thermoautotrophicus in the analogous structural conformations. The resulting structural comparison reveals that thermal adaptation of the M. thermoautotrophicus Fe protein is conferred by a number of factors, including increased structural rigidity that results from various structural changes within the protein interior. The analysis of hypothetical docking models and nitrogenase complex structures provides insights into the thermal adaptation of the protein-protein interactions that support macromolecular complex formation and catalysis at higher temperatures. 相似文献
14.
用对氯汞苯甲酸或二巯基双二硝基苯甲酸修饰巯基。用三硝基苯磺酸、重氮四唑和焦碳酸二乙酯分别修饰氨基、酚式羟基和眯唑基。在一定范围内,被修饰钼铁蛋白在特征波长吸收值的上升和重组活性的下降与修饰剂用量的增加和时间的延长相一致,超过该范围后则光吸收和活性都保持不变。由于无氧凝胶过滤去除多余的修饰剂时未加连二亚硫酸钠,甚至正常钼铁蛋白的重组活性下降了24.2%,修饰后的铝铁蛋白活性下降范围为23.4~41.4%不等。 相似文献
15.
Jason Christiansen Jeannine M. Chan Lance C. Seefeldt Dennis R. Dean 《Journal of inorganic biochemistry》2000,80(3-4)
Site-directed mutagenesis and gene-replacement techniques were used to substitute alanine for the MoFe protein α- and β-subunit phenylalanine-125 residues both separately and in combination. These residues are located on the surface of the MoFe protein near the pseudosymmetric axis of symmetry between the α- and β-subunits. Altered MoFe proteins that contain an alanine substitution at only one of the respective positions exhibit proton reduction activities of about 25–50% when compared to that of the wild-type protein. The lower level of proton reduction also corresponds with decreases in the rates of MgATP hydrolysis. The MoFe protein which contains alanine substitutions in both the α- and β- subunits did not exhibit any proton reduction activity or MgATP hydrolysis. Stopped flow spectrophotometry of the singly substituted MoFe proteins indicate primary electron transfer rate constants approximately an order of magnitude slower than what is observed for wild-type MoFe protein, while no primary electron transfer is observed for the doubly substituted MoFe protein. The doubly substituted MoFe protein is able to interact with the Fe protein as shown by chemical crosslinking experiments. However, this protein does not form a tight complex with the Fe protein when treated with MgADP·AlF4− or when using the altered 127Δ Fe protein. Stopped flow spectrophotometry was also used to quantitate the first-order dissociation rate constants for the two component proteins. These results suggest that the 125Phe residues are involved in an early event(s) that occurs upon component protein docking and could be involved in eliciting MgATP hydrolysis. 相似文献
16.
17.
The assembly of the complex iron-molybdenum cofactor (FeMoco) of nitrogenase molybdenum-iron (MoFe) protein has served as one of the central topics in the field of bioinorganic chemistry for decades. Here we examine the role of a MoFe protein residue (His alpha362) in FeMoco insertion, the final step of FeMoco biosynthesis where FeMoco is incorporated into its binding site in the MoFe protein. Our data from combined metal, activity and electron paramagnetic resonance analyses show that mutations of His alpha362 to small uncharged Ala or negatively charged Asp result in significantly reduced FeMoco accumulation in MoFe protein, indicating that His alpha362 plays a key role in the process of FeMoco insertion. Given the strategic location of His alpha362 at the entry point of the FeMoco insertion funnel, this residue may serve as one of the initial docking points for FeMoco insertion through transient ligand coordination and/or electrostatic interaction. 相似文献
18.
Abstract The effect of certain nitrogen compounds on nitrogenase activity was studied in cells of Azospirillum brasilense strain Sp6, grown under microaerophilic conditions with nitrogenase fully derepressed. 0.5 mM NH4 Cl, 0.5 mM glutamine, 1.0 mM KNO3 and 0.1 mM KNO2 completely blocked nitrogenase activity. 1.0 mM asparagine, 1.0 mM aspartate, 1.0 mM histidine and 1.0 mM adenine did not caused no inhibition of nitrogenase; indeed asparagine, aspartate and histidine showed a slight stimulatory effect on N2 fixation. The addition of 10 mM dl -methionine- dl -sulphoximine prevented the inhibitory effect of NH4 Cl and glutamine but did not counteract the effect of KNO2 . Rifampicin and chloramphenicol did not prevent the inhibition of nitrogenase by NH4 Cl. 相似文献
19.
When the reduced MoFe protein from Azotobacter vinelandii Lipmann was treated with ophenanthroline and air, an inactive protein partially deficient in both FeMoco and P-cluster could be obtained. After incubating the treated protein with a reconstituent solution containing Re2OT, ferric homocitrate, Na2S and dithiothreitol, which had no circular dichroism (CD) signal, the ultraviolet and visible CD spectra, the C2H2 and H+ -reduction activity of the incubated protein were significantly recovered. However, the spectra were somewhat different from those of the reduced MoFe protein. The results showed that: 1) in the incubated protein solution there was possibly a new recombined ReFe protein besides the intact MoFe protein which was not destroyed by the treatment with o-phenanthroline and air; 2) it might be possible that both ReFe protein and MoFe protein exhibited similar ability of nitrogen fixation, although they were somewhat different in structure. 相似文献
20.
含铼重组液对部分缺失金属原子簇的钼铁蛋白的激活作用 总被引:1,自引:0,他引:1
棕色固氮菌(AzotobactervinelandiiLipmann)固氮酶MoFe蛋白经邻菲啉和空气处理后,成为部分缺失P_cluster和FeMoco的失活蛋白。与由Re2O7、高柠檬酸铁、Na2S和二硫苏糖醇(DTT)组成的无圆二色(CD)谱信号的重组液保温后,保温蛋白对乙炔和质子还原的活性都得以显著恢复;紫外和可见光CD谱虽有明显恢复,但仍与还原MoFe蛋白有所差异。这表明:1)保温的蛋白液中除含有未被邻菲啉等处理而破坏的完整MoFe蛋白外,还可能存在新组装的含Re的固氮酶;2)新组装的ReFe蛋白和MoFe蛋白可能在固氮能力上相似,而在结构上有所差别 相似文献