樟子松外生菌根中NADP-依赖型异柠檬酸脱氢酶的酶学性质研究

对褐环乳牛肝菌-樟子松菌根组织、樟子松根组织及褐环乳牛肝菌纯培养菌丝的异柠檬酸脱氢酶(isocitrate dehydrogenase,IDH)进行纯化和酶学性质鉴定。通过硫酸铵分级沉淀及葡聚糖凝胶层析纯化后的IDH进行SDS-PAGE电泳检测,并进行3种来源酶的酶学性质鉴定。菌根组织、根组织及真菌纯培养菌丝NADP-IDH对NADP+的Km值分别为10.7μmol/L、11.4μmol/L和22.1μmol/L;对异柠檬酸的Km值分别为71.7μmol/L、79.3μmol/L和87.8μmol/L。最适p H分别为8.2、8.0和7.5,略偏碱性。菌根IDH和根IDH的最适反应温度为45℃,真菌IDH的最适反应温度为42℃。3种IDH的活性依赖于不同的二价金属阳离子的存在,Mn2+、Mg2+存在时酶活性最强,Ca2+、Co2+、Cu2+和Zn2+对酶的活性有较强抑制作用。菌根真菌与樟子松形成外生菌根之后异柠檬酸脱氢酶的蛋白含量及酶活力都得到了提高。     

固定化谷氨酸棒杆菌T6—13细胞的酶学性质研究

比较研究了固定化谷氨酸棒杆菌细胞和自然细胞的谷氨酸脱氢酶、异拧檬酸脱氢酶,葡萄糖-6-磷酸脱氢酶的一些性质。最适pH、温度对二者酶促反应速度的影响基本相似;pH、热稳定性固定化细胞高于自然细胞;底物表观米氏常数谷氨酸脱氢酶,异柠檬酸脱氢酶有所增大,而葡萄糖-6-磷酸脱氢酶则有所下降;辅酶表观米氏常数均有所增大。这些是影响固定化细胞应用的主要因素。     

克雷伯氏菌甘油脱氢酶dhaD的克隆表达、纯化及酶学性质研究

以克雷伯氏菌基因组DNA为模板,扩增得到编码甘油脱氢酶（GDH）的基因dhaD,将其克隆到大肠杆菌表达载体pET-28a(+)上,在E.coliBL21（DE3）中诱导表达,利用表达载体pET-28a(+)上的6·His-Tag标记选用Ni柱亲和层析法纯化表达具有活性的甘油脱氢酶(GDH),纯化后比酶活达到156U/mg,纯化倍数达4.6倍,回收率为67.4%。并初步研究了该酶的酶学性质,酶反应的最适pH为11.0,在pH7.0～12.0范围内稳定;酶反应的最适温度为30℃,稳定范围为25～45℃; 酶动力学参数以甘油为底物的Km为0.54 mmol/L, Vmax为0.49 μmol/(mL·min)。     

异柠檬酸脱氢酶2突变或诱导机体产生肉瘤

<正>线粒体内依赖辅酶NADP+的异柠檬酸脱氢酶2(IDH2)在新陈代谢中起重要作用,IDH2催化异柠檬酸脱氢生成α-酮戊二酸(αKG),参与三羧酸循环。而当其发生突变时,IDH2则催化异柠檬酸生成2-羟戊二酸(2HG),并竞争性抑制αKG的DNA-组蛋白脱甲基酶,导致细胞分化功能受损。美国宾夕法尼亚大学Lu等人通过全组基因甲基化水平测定,发现IDH2突变通过甲基化DNA CpG岛产生致癌作用,但     

NAD+-依赖型异柠檬酸脱氢酶的结构和功能研究进展

NAD^ —依赖型异柠檬酸脱氢酶是一个核编码线粒体酶,参与三羧酸循环,负责催化异柠檬酸氧化脱羧成α-酮戊二酸,是循环路径中的限速酶。目前在酶学性质、亚基组成、基因克隆、蛋白组装与转运,以及功能等方面开展了许多研究。本文就这些方面的新进展进行综述。     

粘质沙雷氏菌H3010发酵型D-乳酸脱氢酶基因的克隆表达、纯化及酶学性质研究

通过PCR技术从粘质沙雷氏菌H3010基因组DNA中扩增出该D-乳酸脱氢酶基因,连接至pET-28a（＋）表达载体,转入大肠杆菌BL21（DE3）中进行了重组表达,优化了酶纯化的条件,并对其酶学性质进行初步研究。结果表明,获得的该酶编码基因全长993bp,编码330个氨基酸,大小为37kDa。经优化表达及纯化条件后重组酶纯度可达90％。酶学性质研究发现,该重组酶最适反应温度为60℃,最适酶促反应pH为7．5（O．2mol／L磷酸盐缓冲液）,37℃下测得对底物丙酮酸的动力学参数Km=3．39mmol／L,Vmax=6．87mmol／（mg·min）,对辅酶NADH的动力学参数Km=1．43mmol／L,Vmax=1．61mmo]／（mg·min）。为酶法生产D-乳酸及利用代谢工程构建产D-乳酸的基因工程菌打下基础。     

过量表达异柠檬酸裂解酶对ldh-1谷氨酸棒状杆菌产丁二酸的影响

谷氨酸棒状杆菌SA001是缺失了乳酸脱氢酶基因 (ldhA) 的菌株。为了增加厌氧条件下经异柠檬酸到丁二酸的代谢通量,以提高丁二酸的产量。将来自大肠杆菌Escherichia coli K12的异柠檬酸裂解酶基因导入谷氨酸棒状杆菌SA001 (SA001/pXMJ19-aceA) 中。该菌经0.8 mmol/L的IPTG有氧诱导12 h后,转入厌氧发酵16 h,丁二酸的产量为10.38 g/L,丁二酸的生产强度为0.83 g/(L·h)。与出发菌株比较,异柠檬酸裂解酶的酶活提高了5.8倍,丁二酸的产量提高了48%。结果表明过量表达异柠檬酸裂解酶可以增加由乙醛酸途径流向丁二酸的代谢流。     

糖尿病大鼠肋间肌酶组织化学研究

目的探讨糖尿病早期肋间肌酶组织化学变化。方法应用酶组织化学方法观察糖尿病2周和4周大鼠肋间肌组织脱氢酶、水解酶和氧化酶活性变化。结果糖尿病2周大鼠肋间肌组织琥珀酸脱氢酶、谷氨酸脱氢酶和辅酶Ⅰ黄递酶活性较对照组增强,乳酸脱氢酶活性较对照组减弱,苹果酸脱氢酶、异柠檬酸脱氢酶、葡萄糖-6-磷酸脱氢酶、酸性磷酸酶、酸性-α-萘酸性酯酶和细胞色素氧化酶无变化。糖尿病4周大鼠肋间肌组织琥珀酸脱氢酶、苹果酸脱氢酶、谷氨酸脱氢酶、辅酶Ⅰ黄递酶、酸性磷酸酶和酸性-α-萘酸性酯酶活性较对照组增强,乳酸脱氢酶和细胞色素氧化酶活性较对照组减弱,异柠檬酸脱氢酶、葡萄糖-6-磷酸脱氢酶无变化。结论糖尿病2周大鼠肋间肌组织有氧氧化代谢能力增强,糖酵解能力减弱。糖尿病4周大鼠肋间肌组织有氧氧化能力增强、糖酵解能力减弱及能量代谢紊乱。在糖尿病早期呼吸肌存在代谢异常。     

极耐热性乳酸脱氢酶高效表达、纯化及酶学性质

从海栖热袍菌扩增出编码乳酸脱氢酶的基因并将其插入热激载体pHsh构建表达质粒,在大肠杆菌Escherichia coli中进行表达产生极耐热性乳酸脱氢酶Tm-LDH。基因表达产物通过热处理,可以一步获得接近电泳纯的重组酶。酶学性质研究表明,Tm-LDH的最适反应温度为95℃,最适pH 7.0;纯酶在90℃的半衰期为2 h,在pH 5.5–8.0之间最稳定;SDS-PAGE结果显示分子量为33 kDa,与理论推算值相吻合。以丙酮酸和NADH为底物时,相对于丙酮酸的Km值1.7 mmol/L,Vmax为3.8×104 U/mg;相对于NADH的Km值7.2 mmol/L,Vmax值为1.1×105 U/mg。Tm-LDH基因在T7载体中未能实现高效表达,但是在热激载体pHsh中得到了可溶性超量表达,表达水平达到340 mg/L。该酶在65℃反应条件下,活性达到最高活性的50%,并能保持活性不变,这使该酶能够与常温酶匹配,在辅酶NAD再生体系的建立中具有广泛的用途。     

毛栓菌胞外漆酶的纯化及部分性质研究

毛栓菌胞外漆酶经盐析、透析、sephadexG75和G2 5四步纯化 ,粗酶液被纯化了 39 1倍 ,比活力 12 152 ,回收率 4 5 3%。漆酶最适pH值为 4 0 ,最适反应温度为 30℃。K Cu 2 、Zn2 离子可激活漆酶 ,而Ag 、Fe3 离子可抑制漆酶的活性。漆酶的Km值为 1 81× 10 3mol/L。     

Bacillus subtilis isocitrate dehydrogenase. A substrate analogue for Escherichia coli isocitrate dehydrogenase kinase/phosphatase.

In Escherichia coli, the homodimeric Krebs cycle enzyme isocitrate dehydrogenase (EcIDH) is regulated by reversible phosphorylation of a sequestered active site serine. The phosphorylation cycle is catalyzed by a bifunctional protein, IDH kinase/phosphatase (IDH-K/P). To better understand the nature of the interaction between EcIDH and IDH-K/P, we have examined the ability of an IDH homologue from Bacillus subtilis (BsIDH) to serve as a substrate for the kinase and phosphatase activities. BsIDH exhibits extensive sequence and structural similarities with EcIDH, particularly around the phosphorylated serine. Our previous crystallographic analysis revealed that the active site architecture of these two proteins is almost completely conserved. We now expand the comparison to include a number of biochemical properties. Both IDHs display nearly equivalent steady-state kinetic parameters for the dehydrogenase reaction. Both proteins are also phosphorylated by IDH-K/P in the same ratio (1 mole of phosphate per mole of monomer), and this stoichiometric phosphorylation correlates with an equivalent inhibition of IDH activity. Furthermore, tandem electrospray mass spectrometry demonstrates that BsIDH, like EcIDH, is phosphorylated on the corresponding active site serine residue (Ser-104). Despite the high degree of sequence, functional, and structural congruence between these two proteins, BsIDH is surprisingly a much poorer substrate of IDH-K/P than is EcIDH, with Michaelis constants for the kinase and phosphatase activities elevated by 60- and 3,450-fold, respectively. These drastically disparate values might result from restricted access to the active site cavity and/or from the lack of a potential docking site for IDH-K/P.     

Crystal structure of Bacillus subtilis isocitrate dehydrogenase at 1.55 A. Insights into the nature of substrate specificity exhibited by Escherichia coli isocitrate dehydrogenase kinase/phosphatase

Isocitrate dehydrogenase from Bacillus subtilis (BsIDH) is a member of a family of metal-dependent decarboxylating dehydrogenases. Its crystal structure was solved to 1.55 A and detailed comparisons with the homologue from Escherichia coli (EcIDH), the founding member of this family, were made. Although the two IDHs are structurally similar, there are three notable differences between them. First, a mostly nonpolar beta-strand and two connecting loops in the small domain of EcIDH are replaced by two polar alpha-helices in BsIDH. Because of a 13-residue insert in this region of BsIDH, these helices protrude over the active site cleft of the opposing monomer. Second, a coil leading into this cleft, the so-called "phosphorylation" loop, is bent inward in the B. subtilis enzyme, narrowing the entrance to the active site from about 12 to 4 A. Third, although BsIDH is a homodimer, the two unique crystallographic subunits of BsIDH are not structurally identical. The two monomers appear to differ by a domain shift of the large domain relative to the small domain/clasp region, reminiscent of what has been observed in the open/closed conformations of EcIDH. In Escherichia coli, IDH is regulated by reversible phosphorylation by the bifunctional enzyme IDH kinase/phosphatase (IDH-K/P). The site of phosphorylation is Ser(113), which lies deep within the active site crevice. Structural differences between EcIDH and BsIDH may explain disparities in their abilities to act as substrates for IDH-K/P.     

Isocitrate dehydrogenase from the hyperthermophile Aeropyrum pernix: X-ray structure analysis of a ternary enzyme-substrate complex and thermal stability

Isocitrate dehydrogenase from Aeropyrum pernix (ApIDH) is a homodimeric enzyme that belongs to the beta-decarboxylating dehydrogenase family and is the most thermostable IDH identified. It catalyzes the NADP+ and metal-dependent oxidative decarboxylation of isocitrate to alpha-ketoglutarate. We have solved the crystal structures of a native ApIDH at 2.2 A, a pseudo-native ApIDH at 2.1 A, and of ApIDH in complex with NADP+, Ca2+ and d-isocitrate at 2.3 A. The pseudo-native ApIDH is in complex with etheno-NADP+ which was located at the surface instead of in the active site revealing a novel adenine-nucleotide binding site in ApIDH. The native and the pseudo-native ApIDHs were found in an open conformation, whereas one of the subunits of the ternary complex was closed upon substrate binding. The closed subunit showed a domain rotation of 19 degrees compared to the open subunit. The binding of isocitrate in the closed subunit was identical with that of the binary complex of porcine mitochondrial IDH, whereas the binding of NADP+ was similar to that of the ternary complex of IDH from Escherichiacoli. The reaction mechanism is likely to be conserved in the different IDHs. A proton relay chain involving at least five solvent molecules, the 5'-phosphate group of the nicotinamide-ribose and a coupled lysine-tyrosine pair in the active site, is postulated as essential in both the initial and the final steps of the catalytic reaction of IDH. ApIDH was found to be highly homologous to the mesophilic IDHs and was subjected to a comparative analysis in order to find differences that could explain the large difference in thermostability. Mutational studies revealed that a disulfide bond at the N terminus and a seven-membered inter-domain ionic network at the surface are major determinants for the higher thermostability of ApIDH compared to EcIDH. Furthermore, the total number of ion pairs was dramatically higher in ApIDH compared to the mesophilic IDHs if a cutoff of 4.2 A was used. A calculated net charge of only +1 compared to -19 and -25 in EcIDH and BsIDH, respectively, suggested a high degree of electrostatic optimization, which is known to be an important determinant for increased thermostability.     

A highly specific monomeric isocitrate dehydrogenase from Corynebacterium glutamicum

The monomeric isocitrate dehydrogenase (IDH) of Corynebacterium glutamicum is compared to the topologically distinct dimeric IDH of Escherichia coli. Both IDHs have evolved to efficiently catalyze identical reactions with similar pH optimum as well as striking specificity toward NADP and isocitrate. However, the monomeric IDH is 10-fold more active (calculated as kcat/Km.isocitrate/Km.NADP) and 7-fold more NADP-specific than the dimeric enzyme, favoring NADP over NAD by a factor of 50,000. Such an extraordinary coenzyme specificity is not rivaled by any other characterized dehydrogenases. In addition, the monomeric enzyme is 10-fold more specific for isocitrate. The spectacular substrate specificity may be predominantly attributed to the isocitrate-assisted stabilization of catalytic complex during hydride transfer. No significant overall sequence identity is found between the monomeric and dimeric enzymes. However, structure-based alignment leads to the identification of three regions in the monomeric enzyme that match closely the three motifs located in the central region of dimeric IDHs and the homologous isopropylmalate dehydrogenases. The role of Lys253 as catalytic residue has been demonstrated by site-directed mutagenesis. Our results suggest that monomeric and dimeric forms of IDHs are functionally and structurally homologous.     

VEGF165对血管内皮细胞内Mg^2＋浓度调节机制的研究

目的：探讨血管内皮生长因子165（VEGF165）对人脐带静脉内皮细胞（HLNECs）内游离镁离子浓度（[Mg^2＋]i）的调节机制.方法：采用荧光指示剂mag-fura-2及运用PTi阳离子测定系统动态检测HUVECs的[Mg^2＋].结果：经酪氨酸激酶阻断剂（tryrphostin A23和genistein）,磷脂酰3激酶阻断剂（wortmannin和LY294002）,磷脂酶Cγ阻断剂（U73122）预处理,均显著阻断VEGF165诱导的[Mg^2＋]i增加.但经磷脂酶C阻断剂无活性的类似物（U73343）和增殖激活蛋白激酶阻断剂（SB202190和PD9S059）预处理,不能阻断VEGF165诱导的[Mg^2＋]i增加：结论：VEGF165通过酪氨酸激酶／磷脂酰3激酶／磷脂酶口信号转导途径使细胞内的Mg^2＋库释和Mg^2＋,从而增加HUVECs的[Mg^2＋]i.     

Substrate-free structure of a monomeric NADP isocitrate dehydrogenase: an open conformation phylogenetic relationship of isocitrate dehydrogenase

Both monomeric and dimeric NADP+-dependent isocitrate dehydrogenase (IDH) belong to the metal-dependent beta-decarboxylating dehydrogenase family and catalyze the oxidative decarboxylation from 2R,3S-isocitrate to yield 2-oxoglutarate, CO2, and NADPH. It is important to solve the structures of IDHs from various species to correlate with its function and evolutionary significance. So far, only two crystal structures of substrate/cofactor-bound (isocitrate/NADP) NADP+-dependent monomeric IDH from Azotobacter vinelandii (AvIDH) have been solved. Herein, we report for the first time the substrate/cofactor-free structure of a monomeric NADP+-dependent IDH from Corynebacterium glutamicum (CgIDH) in the presence of Mg2+. The 1.75 A structure of CgIDH-Mg2+ showed a distinct open conformation in contrast to the closed conformation of AvIDH-isocitrate/NADP+ complexes. Fluorescence studies on CgIDH in the presence of isocitrate/or NADP+ suggest the presence of low energy barrier conformers. In CgIDH, the amino acid residues corresponding to the Escherichia coli IDH phosphorylation-loop are alpha-helical compared with the more flexible random-coil region in the E. coli protein where IDH activation is controlled by phosphorylation. This more structured region supports the idea that activation of CgIDH is not controlled by phosphorylation. Monomeric NADP+-specific IDHs have been identified from about 50 different bacterial species, such as proteobacteria, actinobacteria, and planctomycetes, whereas, dimeric NADP+-dependent IDHs are diversified in both prokaryotes and eukaryotes. We have constructed a phylogenetic tree based on amino acid sequences of all bacterial monomeric NADP+-dependent IDHs and also another one with specifically chosen species which either contains both monomeric and dimeric NADP+-dependent IDHs or have monomeric NADP+-dependent, as well as NAD+-dependent IDHs. This is done to examine evolutionary relationships.     

Crystal structure of the monomeric isocitrate dehydrogenase in the presence of NADP+: insight into the cofactor recognition,catalysis, and evolution

NADP+-dependent monomeric isocitrate dehydrogenase (IDH) from the nitrogen-fixing bacterium Azotobacter vinelandii (AvIDH) is one of members of the beta-decarboxylating dehydrogenase family and catalyzes the dehydration and decarboxylation of isocitrate to yield 2-oxoglutrate and CO2 in the Krebs cycle. We solved the crystal structure of the AvIDH in complex with cofactor NADP+ (AvIDH-NADP+ complex). The final refined model shows the closed form that has never been detected in any previously solved structures of beta-decarboxylating dehydrogenases. The structure also reveals all of the residues that interact with NADP+. The structure-based sequence alignment reveals that these residues were not conserved in any other dimeric NADP+-dependent IDHs. Therefore the NADP+ specificity of the monomeric and dimeric IDHs was independently acquired through the evolutional process. The AvIDH was known to show an exceptionally high turnover rate. The structure of the AvIDH-NADP+ complex indicates that one loop, which is not present in the Escherichia coli IDHs, reliably stabilizes the conformation of the nicotinamide mononucleotide of the bound NADP+ by forming a few hydrogen bonds, and such interactions are considered to be important for the monomeric enzyme to initiate the hydride transfer reaction immediately. Finally, the structure of the AvIDH is compared with that of other dimeric NADP-IDHs. Several structural features demonstrate that the monomeric IDHs are structurally more related to the eukaryotic dimeric IDHs than to the bacterial dimeric IDHs.     

镁离子对黏液型铜绿假单胞菌生物膜形成过程的影响

目的探讨镁离子对黏液型铜绿假单胞菌早期黏附和生物膜形成过程的影响。方法荧光多功能酶标仪检测各组不同时间点96孔板底部黏附细菌的荧光强度,荧光探针FTTC-ConA染细菌胞外多糖（Extracellular Polymeric Substances,EPS）、荧光显微镜下观察各组多糖差别;SYTO9／PI染生物膜内细菌、激光共聚焦显微镜观察结合BF图像结构分析软件（Image Structor Analyzer,ISA）对各组生物膜结构参数进行定量分析。结果2d时,空白组和1mmol／L镁组的黏附细菌的荧光强度分别为1845．67±45．3和2254．78±42．45,t=-9．96,P〈0．05;0．1mmol／L的镁浓度下荧光强度也有增加,其余各时间组趋势与2d组相似;在荧光显微镜下观察可见随着镁浓度增加,EPS增多;激光共聚焦显微镜下可见随着镁浓度增加,生物膜活菌增加、菌落变密集;ISA软件分析结果示：空白组和1mmol／L镁组的6d生物膜厚度分别为（25．80±1．16）μm和（34．87±1．59）μm,t=-13．85,P〈0．05;区域孔率分别为0．96±0．05和0．90±0．04,t=2．48,P〈0．05;平均扩散距离分别为1．54±0．15和1．92±0．16,t=5．23,P〈0．05;结构熵分别为3．64±0．57和4．70±1．09,t=-2．6,P〈0．05,3d组生物膜也有相同的趋势。结论镁离子可以增强黏液型铜绿假单胞菌的早期黏附,影响随后生物膜的形成及结构。     

植物NADP^＋-依赖型异柠檬酸脱氢酶的分子进化及功能研究进展

高等植物NADP^＋-依赖型异柠檬酸脱氢酶（ICDHs）定位于细胞质、线粒体、叶绿体和过氧化物酶体等植物细胞的不同部位,由不同的基因编码,属于一个高度保守的多同工酶蛋白家族。对近年来关于植物NADP^＋-依赖型异柠檬酸脱氢酶的分子进化及功能研究进行综述,同时提出了未来植物ICDHs的研究重点和方向。最新的分子系统学分析显示,植物中不同细胞定位的ICDH同工酶聚在各自相应的进化枝上,动物或植物中不同细胞器的ICDH同工酶均来源于各自祖先ICDH基因的独立倍增。最新的功能研究表明,ICDHs催化合成的α-酮戊二酸可为植物细胞对氨的吸收同化提供碳骨架,而NADPH可以维系细胞内的氧化还原平衡,帮助植物抵御氧化胁迫。     

产中性纤维素酶耐碱芽孢杆菌的筛选及酶学性质研究

采用CMC碱性平板筛选方法,从造纸厂碱性淤泥中获得产中性纤维素酶的耐碱枯草芽孢杆菌C3004。根据其形态特征、生理生化特性和16S rRNA序列分析鉴定该菌株为枯草芽孢杆菌,并命名为Bacillus subtilis C3004。液体摇瓶培养24 h产生CMC酶活力达46.6 U/mL。酶学性质初步研究显示,CMC酶反应的pH值以7.0左右为适;在弱酸和碱性条件下也具有较高的酶活和一定的稳定性;反应温度以50℃左右为宜;且具有较好的热稳定性。Mn~(2+)与Fe~(3+)对酶反应有促进作用,Cu~(2+)、Mg~(2+)和Zn~(2+)对酶反应有抑制作用。该菌可在碱性(pH 8.5～10)条件下培养,具有不易被杂菌污染的特点。