通过定点突变提高纳豆激酶纤溶活性和热稳定性

纳豆激酶（Nattokinase, NK）是一种纤溶酶，可溶解血栓，常用于治疗心血管疾病（CVDs）。然而，野生型NK往往表现出很少的纤溶能力和较低的热稳定性，针对如何提高NK的热稳定性和活性开展研究。使用重叠延伸PCR法将NK中位于194位的Ser替换为Pro，为验证突变后的热稳定性，将野生型NK和突变体S194P均在不同温度下孵育相同时间，使用纤维蛋白板法测定二者酶活，验证热稳定性。成功构建了pET-26b-NK_S194P，测量酶活结果显示，野生型NK和突变体S194P酶活分别达到101.30 IU/mL和123.23 IU/mL，结果表明突变体S194P的酶活比野生型NK高（18.10±2）%，将野生型NK和突变体S194P在60~65 ℃温度下孵育30 min后测量酶活，野生型NK在63 ℃时丧失酶活，突变体S194P在65 ℃时丧失酶活，耐热温度提高2 ℃。结果表明，突变体S194p的蛋白结构在突变后发生了改变，使NK提高了耐热温度。     

Site-Directed Mutagenesis of Stahphylococcal Nuclease: Role of Tyrosine Residues in Substrate Binding and Catalysis

Abstract

Oligonucleotide directed mutagenesis was used to change specific aminoacid residues in the active site of Staphylococcal nuclease. From the determined kinetic parameters we have calculated the interaction energy of the Tyr side chain with the substrate nitropheny1-pTp.     

The Role for Glutamic Acid at Position 196 in Human Hypoxanthine Phosphoribosyltransferase (HPRT) as Investigated Using Site-Directed Mutagenesis

The crystal structure of human HPRT reveals the involvement of E196 side chain at the A-B dimer interface. Interference by valine substitution at this position (E196V), as identified in patients with Lesch-Nyhan disease, nearly abolishes enzymatic activity. Kinetic analysis of the active mutants (E196A, E196D, E196Q, and E196R) suggests that interaction between K68 and E196 side chains contributes to stabilization of cis-configuration during the catalytic cycle. The study also provides further insight into the role of A-B dimer interactions relating to K68 in the regulation of cis-trans isomerization that potentially governs the rate-limiting steps in the HPRT reaction.     

Significance of the Conserved Tyr352 and Asp380 Residues in the Catalytic Activity of Bacillus stearothermophilus Aminopeptidase II as Evaluated by Site-directed Mutagenesis

The importance of the conserved Tyr352 and Asp380 residues of Bacillus stearothermophilus aminopeptidase II (AP-II) was investigated by site-directed mutagenesis. The wild-type and mutant enzymes were expressed in recombinant Escherichia coli M15 cells and the 45-kD proteins were purified from the cell-free extracts by Ni(2+)-NTA resin. The specific activity for Tyr352 and Asp380 replacements was decreased by more than 3.5-fold. Detailed analysis of the kinetic consequences in the mutant proteins revealed that the K (m) values were increased 1.9- to 2.6-fold with respect to wild-type enzyme. Catalytic efficiencies (k (cat)/K (m)) of mutant proteins were between 3.5- and 31-fold lower than the corresponding value of the wild-type enzyme. Tryptophan emission fluorescence and circular dichroism spectra were nearly identical for wild-type and mutant enzymes. These results indicate that residues Tyr352 and Asp380 are essential for the proper function of AP-II.     

The Role of the Disulfide Bridge in the Stability and Structural Integrity of Ovalbumin Evaluated by Site-Directed Mutagenesis

To provide a molecular explanation of the role of the disulfide (SS) bridge in the thermostability and structural integrity of ovalbumin (OVA), we prepared SS-mutated OVAs in which SS-forming residues were replaced by Ala or Ser (C73A, C73S, C120A, and C73/120A), and compared the conformation, thermostability, susceptibility to elastase, and formation of heat-stable OVA (S-OVA) with those of the wild-type. The circular dichroism (CD) and tryptophan fluorescence spectra revealed that the SS-mutated OVAs assumed a native-like conformation similar to the wild-type. The thermal denaturation temperature for the SS-mutated OVAs was significantly lower than that for the wild-type. C73S, C120A, and C73/120A mutants converted to S-OVA on alkaline treatment. Analyses for elastase digestion fragments showed that a non-native SS bridge was generated in all SS-mutated OVAs, but non-native SS-pairing did not contribute to thermostability. Hence, we concluded that the presence of the original SS bridge in OVA contributes to conformational stability but is not directly related to the conversion to S-OVA.     

Enhancement of the Alcoholytic Activity of α-Amylase AmyA from Thermotoga maritima MSB8 (DSM 3109) by Site-Directed Mutagenesis

AmyA, an α-amylase from the hyperthermophilic bacterium Thermotoga maritima, is able to hydrolyze internal α-1,4-glycosidic bonds in various α-glucans at 85°C as the optimal temperature. Like other glycoside hydrolases, AmyA also catalyzes transglycosylation reactions, particularly when oligosaccharides are used as substrates. It was found that when methanol or butanol was used as the nucleophile instead of water, AmyA was able to catalyze alcoholysis reactions. This capability has been evaluated in the past for some α-amylases, with the finding that only the saccharifying fungal amylases from Aspergillus niger and from Aspergillus oryzae present measurable alcoholysis activity (R. I. Santamaria, G. Del Rio, G. Saab, M. E. Rodriguez, X. Soberon, and A. Lopez, FEBS Lett. 452:346-350, 1999). In the present work, we found that AmyA generates larger quantities of alkyl glycosides than any amylase reported so far. In order to increase the alcoholytic activity observed in AmyA, several residues were identified and mutated based on previous analogous positions in amylases, defining the polarity and geometry of the active site. Replacement of residue His222 by glutamine generated an increase in the alkyl glucoside yield as a consequence of a higher alcoholysis/hydrolysis ratio. The same change in specificity was observed for the mutants H222E and H222D, but instability of these mutants toward alcohols decreased the yield of alkyl glucoside.     

Purification, characterization and cDNA cloning of an analgesic peptide from the Chinese scorpion Buthus martensii Karsch (BmK AGP-SYPU2)

In this study an analgesic peptide was purified through five continuous chromatographic steps. The mouse twisting model test was used to identify the target peptides in every separation step. The purified BmK AGP-SYPU2 was further qualified by Reverse Phase-High Performance Liquid Chromatography and High Performance Capillary Electrophoresis. The molecular weight, isoelectric point, and N-terminal sequence of the purified peptide were determined. Based on the N-terminal sequence, the cDNA was cloned by rapid amplification of the cDNA ends from the cDNA pool of scorpion glands. Sequence determination showed that the mature BmK AGP-SYPU2 peptide is composed of 66 amino acid residues, and BmK AGP-SYPU2 is identical to BmK alpha2 (GenBank Acc. No. AF288608) and BmK alphaTX11 (GenBank Acc. No. AF155364). We report herein a purification procedure that yields substantial amounts of natural BmK AGP-SYPU2 with high analgesic activity.     

Investigation of the Role of Conserved Serine Residues in the Long Form of the Rat D2 Dopamine Receptor Using Site-Directed Mutagenesis

Abstract: Three serine residues (Ser¹⁹³, Ser¹⁹⁴, Ser¹⁹⁷) in the fifth transmembrane-spanning region of the D₂ dopamine receptor have been mutated separately to alanine and the effects of the mutations determined in ligand-binding experiments with [³H]spiperone. For many antagonists the mutations had little effect, showing that the overall conformation of the mutant receptors was similar to that of the native, although there were effects on the binding of certain antagonists. The effect of the mutations on agonist binding to the free receptor (uncoupled from G proteins) was determined in the presence of GTP (100 µ M ). This showed that there was no single mode of binding of catecholamine agonists to the receptor and that all three serine residues can participate in the binding of some agonists, possibly through hydrogen bonds to the catechol hydroxyl groups. Coupling of the mutant receptors to G proteins was assessed from agonist-binding curves in the absence of GTP, when higher and lower affinity agonist-binding sites were seen. Receptor/G protein coupling was generally unaffected by the Ala¹⁹³ and Ala¹⁹⁴ mutations, but the Ala¹⁹⁷ mutation eliminated receptor/G protein coupling for some agonists. These data show that the interactions of agonists with the free and coupled forms of the receptor are different.     

Role of Serine-19 Phosphorylation in Regulating Tyrosine Hydroxylase Studied with Site- and Phosphospecific Antibodies and Site-Directed Mutagenesis

Abstract: The effects of depolarization by elevated potassium concentrations were studied in PC12 cells and in stably transfected AtT-20 cells expressing wild-type or [Leu¹⁹]-recombinant tyrosine hydroxylase (rTH). Changes in the phosphorylation states of Ser¹⁹ and Ser⁴⁰ in tyrosine hydroxylase (TH) were determined immunochemically using antibodies specific for the phosphorylated state of each site and compared with changes in TH activity in PC12 cell lysates and with changes in l -DOPA biosynthesis rates in intact AtT-20 cells. Treatment of either PC12 cells or AtT-20 cells expressing wild-type rTH with elevated potassium produced a transient increase in the phosphorylation state of Ser¹⁹ (up to 0.7 mol of phosphate/mol of subunit) in concert with a more gradual and sustained increase in Ser⁴⁰ phosphorylation. Elevated potassium treatment also increased TH activity in PC12 cell lysates, but these increases paralleled the temporal course of Ser⁴⁰, as opposed to Ser¹⁹, phosphorylation. Similarly, increases in DOPA accumulation produced by elevated potassium in AtT-20 cells expressing wild-type rTH paralleled the increases in the phosphorylation state of Ser⁴⁰ but not Ser¹⁹. Moreover, elevated potassium produced comparable increases in DOPA accumulation in AtT-20 cells expressing rTH in which Ser¹⁹ phosphorylation had been eliminated (by substitution of Leu for Ser¹⁹). Thus, depolarization-induced increases in the stoichiometry of Ser¹⁹ phosphorylation do not appear to influence directly the activity of TH in situ.     

Site-Directed Mutagenesis of the Virion Host Shutoff Gene (UL41) of Herpes Simplex Virus (HSV): Analysis of Functional Differences between HSV Type 1 (HSV-1) and HSV-2 Alleles

During lytic herpes simplex virus (HSV) infections, the HSV virion host shutoff protein (UL41) accelerates the turnover of host and viral mRNAs. Although the UL41 polypeptides from HSV type 1 (HSV-1) strain KOS and HSV-2 strain 333 are 87% identical, HSV-2 strains generally shut off the host more rapidly and completely than HSV-1 strains. In a previous study, we identified three regions of the HSV-2 UL41 polypeptide (amino acids 1 to 135, 208 to 243, and 365 to 492) that enhance the activity of KOS when substituted for the corresponding portions of the KOS protein (D. N. Everly, Jr., and G. S. Read, J. Virol. 71:7157-7166, 1997). These results have been extended through the analysis of more than 50 site-directed mutants of UL41 in which selected HSV-2 amino acids were introduced into an HSV-1 background and HSV-1 amino acids were introduced into the HSV-2 allele. The HSV-2 amino acids R22 and E25 were found to contribute dramatically to the greater activity of the HSV-2 allele, as did the HSV-2 amino acids A396 and S423. The substitution of six HSV-2 amino acids between residues 210 and 242 enhanced the HSV-1 activity to a lesser extent. In most cases, individual substitutions or the substitution of combinations of fewer than all six amino acids reduced the UL41 activity to less than that of KOS. The results pinpoint several type-specific amino acids that are largely responsible for the greater activity of the UL41 polypeptide of HSV-2. In addition, several spontaneous mutations that abolish detectable UL41 activity were identified.     

Functional implications of the proximal site hydrogen bonding network in Vitreoscilla hemoglobin (VHb): role of Tyr95 (G5) and Tyr126 (H12)

Although Vitreoscilla hemoglobin (VHb) carries a conventional globin fold, its proximal site geometry is unique in having a hydrogen-bonding network between proximal site residues, HisF8-TyrG5-GluH23 and TyrG5-TyrH12. TyrG5 and TyrH12 were mutated to study their relevance in VHb function. VHb G5 mutants (Tyr95Phe and Tyr95Leu showed no stable oxyform and nitric oxide dioxygenase activity, whereas, VHb H12 mutants (Tyr126Phe and Tyr126Leu) displayed little change in their oxygen affinity indicating a crucial role of Tyr95 in protein function. The VHb H12 mutant, Tyr126Leu, enhanced the intracellular pool of oxygen and cell growth better than VHb. Molecular modeling suggests that the replacement of tyrosine with leucine in Tyr126Leu creates an opening on the protein surface that may facilitate oxygen diffusion and accumulation.     

Demand-Driven Control of Root ATP Sulfurylase Activity and SO42- Uptake in Intact Canola (The Role of Phloem-Translocated Glutathione)

The activity of ATP sulfurylase extracted from roots of intact canola (Brassica napus L. cv Drakkar) increased after withdrawal of the S source from the nutrient solution and declined after refeeding SO42- to S-starved plants. The rate of SO42- uptake by the roots was similarly influenced. Identical responses were obtained in SO42- -fed roots when one-half of the root system was starved for S. The internal levels of SO42- and glutathione (GSH) declined after S starvation of the whole root system, but only GSH concentration declined in +S roots of plants from split root experiments. The concentration of GSH in phloem exudates decreased upon transfer of plants to S-free solution. Supplying GSH or cysteine to roots, either exogenously or internally via phloem sap, inhibited both ATP sulfurylase activity and SO42- uptake. Buthionine sulfoximine, an inhibitor of GSH synthesis, reversed the inhibitory effect of cysteine on ATP sulfurylase. It is hypothesized that GSH is responsible for mediating the responses to S availability. ATP sulfurylase activity and the SO42- uptake rate are regulated by similar demand-driven processes that involve the translocation of a phloem-transported message (possibly GSH) to the roots that provides information concerning the nutritional status of the leaves.     

隐地蛋白(cryptogein)基因定点突变及其广谱抗病烟草转化植株的获得

用PCR法从隐地疫霉 (Phytophthoracryptogea)基因组DNA中克隆了cryptogein(Cry)基因。将Cry基因的 13位赖氨酸 (K)突变成缬氨酸 (V) ,获突变基因CryK13V ,并将其构建于CaMV35S启动子控制的植物表达载体上。通过农杆菌介导的叶盘转化法转入烟草 ,经卡那霉素抗性筛选获 33株再生植株 ,PCR检测和Southern杂交分析表明CryK13V基因已整合到烟草基因组中。接种试验结果表明 ,转基因烟草植株对黑胫病菌、赤星病菌和野火病菌等的抗性均有提高。Northern杂交分析表明 ,微弱的CryK13V基因在转化植株中的表达就足以激活PR1和OPBP1等防卫反应相关基因的表达 ,而且表达丰度与转基因植株的抗病性有着一定的正相关性。研究结果还表明 ,隐地蛋白13位上的赖氨酸在诱导细胞死亡中起着关键的作用。     

岩白菜素两个衍生物的合成及镇痛活性研究

本文研究两个岩白菜素衍生物的合成及其镇痛活性.以岩白菜素为原料,通过选择性磺酰化和曼尼希反应各自得到一个产物.采用小鼠扭体法检测两个化合物的镇痛活性.两个化合物分别在剂量为60 mg/kg,100 mg/kg时可明显抑制小鼠扭体次数,试药组与生理盐水组比较差异具有显著性意义(P<0.05).对比两衍生物的结构提示镇痛活性可能与岩白菜素的基本骨架有关.     

Defining a Conformational Consensus Motif in Cotransin-Sensitive Signal Sequences: A Proteomic and Site-Directed Mutagenesis Study

The cyclodepsipeptide cotransin was described to inhibit the biosynthesis of a small subset of proteins by a signal sequence-discriminatory mechanism at the Sec61 protein-conducting channel. However, it was not clear how selective cotransin is, i.e. how many proteins are sensitive. Moreover, a consensus motif in signal sequences mediating cotransin sensitivity has yet not been described. To address these questions, we performed a proteomic study using cotransin-treated human hepatocellular carcinoma cells and the stable isotope labelling by amino acids in cell culture technique in combination with quantitative mass spectrometry. We used a saturating concentration of cotransin (30 micromolar) to identify also less-sensitive proteins and to discriminate the latter from completely resistant proteins. We found that the biosynthesis of almost all secreted proteins was cotransin-sensitive under these conditions. In contrast, biosynthesis of the majority of the integral membrane proteins was cotransin-resistant. Cotransin sensitivity of signal sequences was neither related to their length nor to their hydrophobicity. Instead, in the case of signal anchor sequences, we identified for the first time a conformational consensus motif mediating cotransin sensitivity.