EcoRV-T94V: a mutant restriction endonuclease with an altered substrate specificity towards modified oligodexynucleotides

Synthetic oligodeoxynucleotides with single methyl phosphonate(mp) substitutions were used for an analysis of the contributionof phosphate contacts to the recognition of the cleavage siteby the restriction endonuclease EcoRV. Only in the last positionwithin the recognition sequence, is the methyl phosphonate substitutiontolerated by the enzyme. The wild-type enzyme cleaves the S_Pdiastereomer of the oligodeoxynucleotide GACGATAT_mpCGTC andthe unmodified sequence with equal rates, whereas the R_P diastereomeris cleaved much more slowly. Inspection of the crystal structureof an EcoRV–DNA complex revealed that the non-bridgingoxygen atoms of the phosphodiester bond between the T and Cbases are in hydrogen bonding distance of the hydroxyl groupof the amino acid Thr94. We therefore tried to engineer a variantof EcoRV that would prefer a methyl phosphonate linkage overa normal phosphodiester bond and produced mutants with aminoacid exchanges at position 94. One of them, Thr94Val, showsa dramatically reduced activity towards the unmodified DNA anddoes not accept the R_p diastereomer, but cleaves the S_P diastereomerwith the same rate as wild-type EcoRV. Its selectivity, i.e.the ratio of cleavage rates determined for the unmodified andmodified substrates, differs by three orders of magnitude fromthat of the wild-type enzyme.     

The structural basis for the altered substrate specificity of the R292D active site mutant of aspartate aminotransferase from E.coli

Two refined crystal structures of aspartate aminotransferasefrom E.coli are reported. The wild type enzyme is in the pyridoxalphosphate (PLP) form and its structure has been determined to2.4 Å resolution, refined to an R-factor of 23.2%. Thestructure of the Arg292Asp mutant has been determined at 2.8Å resolution, refined to an R–factor of 20.3%. Thewild type and mutant crystals are isomorphous and the two structuresare very similar, with only minor changes in positions of importantactive site residues. As residue Arg292 is primarily responsiblefor the substrate charge specificity in the wild type enzyme,the mutant containing a charge reversal at this position mightbe expected to catalyze transamination of arginine as efficientlyas the wild type enzyme effects transamination of aspartate[Cronin,C.N. and Kirsch,J.F. (1988) Biochemistry, 27, 4572–4579].This mutant does in fact prefer arginine over aspartate as asubstrate, however, the rate of catalysis is much slower thanthat of the wild type enzyme with its physiological substrate,aspartate. A comparison of these two structures indicates thatthe poorer catalytic efficiency of R292D, when presented witharginine, is not due to a gross conformational difference, butis rather a consequence of both small side chain and main chainreorientations and the pre–existing active site polarenvironment, which greatly favors the wild type ion pair interaction.     

Mutational analysis of DNase I-DNA interactions: design, expression and characterization of a DNase I loop insertion mutant with altered sequence selectivity

A mutant of bovine pancreatic DNase I containing two additionalresidues in a loop next to C173 has been expressed in Escherichiacoli, purified and characterized biochemically. Modelling studiessuggest that the inserted arginine and glutamate side chainsof the modified loop sequence C173-R-E-G-T-V176 could contactthe bases 3' to the cleaved bond in the major groove of a boundDNA, and that up to 10 bp could interact with the enzyme andpotentially influence its cutting rate. The loop insertion mutanthas an 800-fold lower specific activity than wild-type and showsoverall cleavage characteristics similar to bovine pancreaticDNase I. Compared with the wild-type enzyme, the mutant showsa strongly enhanced preference for cutting the inverted repeat:5'-GACTT A AAGTC-3' CTGAA T TTCAG or close variants thereof.Unexpectedly for a minor groove binding protein, the preferredcutting sites in opposite strands are staggered by 1 bp in the5' direction, causing the cleavage of a TA and a TT step, respectively.This finding demonstrates that the sequence context is relativelymore important for the cutting frequency than the nature ofthe dinucleotide step of the cleaved bond, and clearly showsthat base recognition is involved in determining the sequenceselectivity of the mutant. The importance of the sequence 5'to the cleaved bond for the cutting rate suggests that the additionalmajor groove contacts may require a distortion of the DNA associatedwith a higher energy barrier, resulting in an increased selectivityfor flexible DNA sequences and a lower overall activity of themutant enzyme.     

3-D structure of a mutant (Asp101 -> Ser) of E.coli alkaline phosphatase with higher catalytic activity

Mutagenesis of the absolutely conserved residue Asp101 of thenon-specific monoesterase alkaline phosphatase (E.C. 3.1.3.1 [EC] )from E.coli has produced an enzyme with increased k_cat. Thecarboxyl group of the Asp101 residue has been proposed to beinvolved in the positioning of Arg166 and the formation of thehelix that contains the active site Ser 102. The crystal structureof the Asp101     

Three-dimensional structure of a mutant E.coli aspartate aminotransferase with increased enzymic activity

The aspartate and tyrosine aminotransferases from Escherichiacoli have 43% sequence identity and nearly identical activesites. Both are equally good enzymes for dicarboxylate substrates,but the latter transaminates aromatic amino acids 1000 timesfaster. In an attempt to discover the critical residues forthis differential substrate specificity, the aspartate aminotransferasemutant V39L has recently been prepared. It showed improved k_cal/K_mvalues for aspartate, glutamate and tyrosine and the correspondingoxo acids, mainly due to two to ten times lower K_m values. Forexample, the K_m values of V39L (wild type) for Asp and Glu are0.12 (1.0) and 0.85 (2.7) mM respectively. The mutant was co-crystallizedwith 30 mM maleate from both polyethylene glycol and ammoniumsulfate. Both structures were solved and refined to R-factorsof 0.22 and 0.20 at 2.85 and 2.5 Å resolution respectively.They bear strong resemblance to the closed structure of thewild type enzyme complexed with maleate. The unexpected featureis that, for the first time, the closed form was produced incrystals grown from ammonium sulfate. It is concluded that themutation has shifted the conformational equilibrium towardsthe closed form, which leads to generally reduced substrateK_mS     

Construction of novel subtilisin E with high specificity, activity and productivity through multiple amino acid substitutions

Through three cumulative amino acid substitutions, we constructed novel mutant subtilisins E of Bacillus subtilis, all with high specificity, activity and productivity. The substitution of conserved Gly127, constituting P1 substrate-binding pocket, with Ala and Val showed a marked preference for the small P1 substrate. Leu was then substituted for Ile31 next to the catalytic Asp32 to enhance the catalytic activity. Both double mutants (I31L/G127A and I31L/G127V) showed a 3-5- fold increase in catalytic efficiency due to a large kcat, without any change in the specificity of the mutants at position 127. Molecular modeling suggests that large P1 residues were unable to access the pocket because of steric hindrance. A third mutation was introduced by replacing Tyr(-1) with Ala in the propeptide essential for autoprocessing to active mature subtilisin in vivo. A prominent 7-20- fold increase in active enzyme production occurred in the triple mutants (Y-1A/I31L/G127A and Y-1A/I31L/G127V).      

Oxidative stability and AromaScan analyses of corn oils with altered fatty acid content

This project was designed to evaluate the oxidative stability of corn oils with increased total saturated fatty acid composition and to test the feasibility of using the AromaScan, an “electronic nose,” to detect the odors/aromas produced by oxidation. Corn oils with traditional (13.1%) and elevated (14.7 to 17.1%) total saturated fatty acid percentages were evaluated for their oxidative quality. Oils from five corn genotypes were extracted, refined, bleached, and deodorized (RBD) in the laboratory. Two replications, separated at the point of extraction, were evaluated for each genotype. The RBD corn oils (18.0 g) were stored in 50-mL beakers at 60°C in the dark, and peroxide values were measured every other day for 8 d. Corn oils with elevated saturated fatty acid compositions were more stable (P<0.05) than the traditional corn oil. Aroma intensity of the oils was measured with an AromaScan at days 0, 4, and 8. The AromaScan provided a useful tool to detect odors/aromas produced by oxidation during an oxidative stability study; this tool might be used to partly replace human sensory panel evaluation of oxidized samples.     

Streptomyces phospholipase D mutants with altered substrate specificity capable of phosphatidylinositol synthesis

The substrate specificity of a phospholipase D (PLD) from Streptomyces antibioticus was altered by site-directed saturation mutagenesis, so that it was able to synthesize phosphatidylinositol (PI). Mutations were introduced in the pld gene at the positions corresponding to three amino acid residues that might be involved in substrate recognition, and the mutated genes were expressed in Escherichia coli BL21 (DE3). High-throughput screening of approximately 10,000 colonies for PI-synthesizing activity identified 25 PI-synthesizing mutant PLDs. One of these mutant enzymes was chosen for further analysis. The structure of the PI synthesized with the mutant enzyme was analyzed by HPLC-MS and NMR. It was found that the mutant enzyme generated a mixture of structural isomers of PIs with the phosphatidyl groups connected at different positions of the inositol ring. The phosphatidylcholine-hydrolyzing activity of the mutant PLD was much lower than that of the wild-type enzyme. The mutant enzyme was able to transphosphatidylate various cyclohexanols with a preference for bulkier compounds. This is the first example of alteration of the substrate specificity of PLD and of PI synthesis by Streptomyces PLD.     

成纤维细胞生长因子受体基因野生型和E731K突变型真核表达质粒的构建及鉴定

目的构建成纤维细胞生长因子受体(fibroblast growth factor receptor 2,FGFR2)基因野生型和E731K突变型的真核表达载体,并进行鉴定。方法利用基因定点突变试剂盒,定点突变FGFR2基因,获得其E731K突变的突变型基因,通过设计含有XbaⅠ、XhoⅠ限制性内切酶识别序列的引物分别扩增FGFR2基因野生型和E731K突变型的cDNA,克隆至质粒pcDNA3.1-EGFP上,构建重组表达质粒pcDNA3.1-FGFR2-EGFP和pcDNA3.1-FGFR2E731K-EGFP,利用X-tremeGENE HP DNA将质粒转染至HEK293细胞,采用实时荧光定量PCR法及Western blot法检测FGFR2表达水平。结果经定点突变已获得野生型的突变型FGFR2基因,碱基序列与设计序列完全一致,cDNA第2191位碱基G突变为A。重组表达质粒pcDNA3.1-FGFR2-EGFP和pcDNA3.1-FGFR2E731K-EGFP经双酶切及测序鉴定证明构建正确。质粒转染HEK293细胞后,荧光显微镜下均可见绿色荧光蛋白表达;质粒pcDNA3.1-FGFR2-EGFP和pcDNA3.1-FGFR2E731K-EGFP转染组中FGFR2 mNRA和蛋白表达水平均明显高于质粒pcDNA3.1-EGFP转染组和空白对照组(P0.05)。结论成功构建了FGFR2基因野生型和E731K突变型的真核表达质粒,并在HEK293细胞中成功表达,为进一步研究FGFR2基因奠定了基础。     

Prediction of higher heating values of biomass from proximate and ultimate analyses

Two new empirical correlations based on proximate and ultimate analyses of biomass used for prediction of higher heating value (HHV) are presented in this paper. The correlations have been developed via stepwise linear regression method by using data of biomass samples (from the open literature) of varied origin and obtained from different geographical locations. The correlations have been validated via incorporation of additional biomass data. The correlation based on ultimate analysis (HHV = 0.2949C + 0.8250H) has a mean absolute error (MAE) lower than 5% and marginal mean bias error (MBE) at just 0.57% which indicate that it has good HHV predictive capability. The other correlation which is based on proximate analysis (HHV = 0.1905VM + 0.2521FC) is a useful companion correlation with low absolute MBE (0.67%). The HHV prediction accuracies of 12 other correlations introduced by other researchers are also compared in this study.     

Engineering subtilisin YaB: restriction of substrate specificity by the substitution of Gly124 and Gly151 with Ala

The 3-D structure of subtilisin YaB was computer modelled using thestructures of subtilisin BPN', subtilisin Carlsberg and thermitase astemplates. Gly124 and Gly151 located on both sides of the waist of the S1pocket were selected for site-directed mutagenesis based on the modelledstructure. The mutated ale genes coding for the mutant subtilisin YaB wereexpressed in Bacillus subtilis DB104. All of the G124 and G151 series ofmutants exhibited an increase of relative catalytic activity forelastin-orcein against casein and myofibrillar proteins. The S1 substratespecificity of G124A, G124V and G151A mutants were assessed using variouscarbobenzoxy-amino acid-nitrophenyl esters and succinyl-Ala-Ala-(Pro orVal)-(Ala, Phe or Leu)-p- nitroanilide [AA(P/V) (A/F/L)]. While G124A andG124V mutants hydrolyzed only Ala and Gly esters, G151A mutant hydrolyzedAla, Leu and Gly esters. The G124A and G124V mutants did not hydrolyze AAPFand AAPL. However, these two mutants hydrolyzed AAPA and AAVA with kcat/Kmvalues approximately 3-10-fold higher than those of the wild-type enzyme.The G151A mutant did not hydrolyze AAPF, but hydrolyzed AAPL, AAPA and AAVAwith kcat/Km values approximately 1-4-fold higher than those of thewild-type enzyme. These results clearly indicate that the S1 substratespecificity of G124A and G124V mutants was restricted to Ala and Gly, andG151A mutant to Ala, Gly and Leu.     

Evolution of interleukin-15 for higher E. coli expression and solubility

Directed evolution was used to generate IL-15 mutants with increased solubility and cytoplasmic over-expression in Escherichia coli. A protein solubility selection method was used in which the IL-15 gene was expressed as an N-terminal fusion to chloramphenicol acetyltransferase (CAT) as reporter protein. Clones that grew in the presence of high concentrations of chloramphenicol were then screened by ELISA to assay the binding activity of the IL-15-CAT fusion to the IL-15Rα Sushi domain. Two variants of IL-15, M38 and M253, containing five mutations and one mutation respectively, were selected with a dramatic improvement in solubility; the soluble concentration in cell culture was 12- to 18-fold higher, respectively, than for WT IL-15. Characterization of their binding to IL-15Rα and their ability to stimulate the T-cell growth response showed that M38 binds as strongly as native IL-15 to IL-15Rα and acts as an effective agonist of IL-15.     

Mutational and structural analysis of the lectin activity in binding domain 2 of ricin B chain

The study of the lectin binding sites of ricin B chain and ofother homologous members of the small gene family that makeup ricin-like molecules has revealed a number of key contactresidues involved in sugar binding. In particular, on the basisof data generated by the X-ray crystallographic structure ofricin, comparisons of sequence homologies to other ricin-likemolecules and substrate binding studies with these molecules,it has been proposed that His248 of Ricinus communis agglutinin(RCA) B chain may interfere with galactose binding in the secondbinding domain of that lectin. To test that hypothesis, singlebinding domain 2 (SBD2) of ricin B chain was expressed as agene 3 fusion protein on the surface of fd phage to measuredirectly the effect of mutational changes on this binding site.Replacement of tyrosine with histidine at amino acid position248 of SBD2 of ricin B chain was shown to reduce lectin activity.The sequences of RCA and ricin B chains were aligned and comparedwith the tertiary structure of ricin B chain to select variousmutations that were introduced as controls in the study. Oneof these controls, Leu247 to Val247, displayed increased affinityfor galactosides. The role of sequence changes is discussedin relation to the structural and functional divergence in thesemolecules.     

Characterization and peroxidase activity of a myoglobin mutant containing a distal arginine

The spectroscopic, conformational, and reactivity characteristics of the T67R variant of sperm whale myoglobin have been studied to assess the effects of introducing an arginine residue into the distal side of this protein, as occurs in the active site of heme peroxidases. The overall circular dichroism (CD) and NMR spectroscopic properties of various derivatives of the protein are little affected by the mutation. The mutant contains a high-spin ferric ion with a water molecule as the sixth ligand, which exhibits slightly enhanced acidity (pK(a)=8.43+/-0.03) with respect to the corresponding derivative of wild-type myoglobin (pK(a)=8.60+/-0.04). The presence of the distal arginine increases the affinity of the Fe(III) center for azide (K=(6.0+/-0.5)x10(4) M(-1)) and decreases that for imidazole (K=12.0+/-0.2 M(-1)), with respect to the wild-type protein (K=(5.0+/-0.1)x10(4) and 24.7+/-0.7 M(-1), respectively). The peroxidase activity of T67R and wild-type myoglobins has been studied with a group of phenolic substrates related to tyrosine. The mutant exhibits an increased rate of reaction with hydrogen peroxide (k=1550+/-10 versus 760+/-10 M(-1) x s(-1)) and a generally increased peroxidase activity with respect to wild-type myoglobin. Relaxation measurements of proton nuclei of the phenolic substrates in the presence of either the T67R variant or the wild-type protein show that binding of these molecules occurs at distances of 8-10 A from the iron center, that is, close to the heme pocket, except for p-cresol, which can approach the heme more closely and, therefore, probably enter into the distal cavity.     

Mutational analysis of human glutathione transferase A2-2 identifies structural elements supporting high activity with the prodrug azathioprine

Glutathione transferase (GST) A2-2 is the human enzyme displaying the highest catalytic activity with the prodrug azathioprine (Aza). The reaction releases pharmacologically active 6-mercaptopurine by displacing the imidazole moiety from the Aza molecule. The GST-catalyzed reaction is of medical significance, since high rates of Aza activation may lead to adverse side effects in treated patients. The present study involves structure-activity relationships in GST A2-2 variants. Chimeric GSTs were previously generated by DNA shuffling and two peptide segments, one N-terminal and one C-terminal, were identified as primary determinants of Aza activity. The segments contain several residues of the substrate-binding H-site and their significance for supporting high Aza activity was investigated. Substitution of the corresponding two small regions in the low-activity human GST A3-3 or rat GST A3-3 by the human GST A2-2 segments generated chimeras with ～10-fold enhanced Aza activity. The H-site residues Met208 and Leu213 in the C-terminal segment of GST A2-2 were mutated to produce a library with all possible residue combinations. At a calculated 93% library coverage, all of the 1880 mutants examined showed wild-type or decreased Aza activity, even though some retained activities with alternative substrates, further emphasizing the importance of this region for the targeted activity.     

A novel bis-aziridinylnaphthoquinone with anti-solid tumor activity in which induced apoptosis is associated with altered expression of Bcl-2 protein

Aziridine-containing compounds have been of interest as anticancer agents since the late 1970s. The design, synthesis, and study of aziridinylnaphthoquinone analogues to obtain compounds with enhanced activity/toxicity profiles are an ongoing research effort in our group. A series of bis-aziridinylnaphthoquinone derivatives has been prepared, and the cytotoxic activities of these synthetic bis-aziridinylnaphthoquinone derivatives has been investigated. The synthetic derivatives displayed significant cytotoxicity against human carcinoma cell lines and weak cytotoxic activities against skin fibroblasts (SF). The bis-aziridinylnaphthoquinone 1 c was the most effective of the tested analogues at reducing the viability of Hep2 cells, with an LD(50) value of 5.23 microM, and also exhibited weak cytotoxic activity against SF cells, with an LD(50) value of 54.12 microM. The DNA alkylation and DNA interstrand cross-linking abilities of 1 c were also investigated. Bis-aziridinylnaphthoquinone 1 c was an effective agent for alkylation of DNA after chemical reduction in vitro, and its bifunctional alkylating moieties were able to cross-link DNA. We also report here our efforts to determine direct antitumor effects of 1 c on Hep2 cells. Growth arrest in Hep2 cells was preceded by early induction of G(2)-M cell cycle arrest at 0.75 microM of 1 c after culture for 24 h, and was then followed by apoptosis after 60 h. This was associated with decreased expression of antiapoptotic bcl2 protein (by 78 %) upon culture with 3.0 microM of 1 c after 60 h. Our results suggest that 1 c is a novel antitumor aziridinylnaphthoquinone with therapeutic potential against solid tumors.     

Novel mutant human fibronectin FIII9-10 domain pair with increased conformational stability and biological activity

The ninth and tenth type III domains (FIII9–10) in thecentral cell binding domain of human fibronectin contain integrinreceptor binding sites, including RGD in FIII10 and a synergysite, PHSRN, in FIII9. The specific amino acids that contributeto cell binding have been identified by the use of wild-typeand mutant fragments of human fibronectin containing the FIII9–10domain pair. At high concentrations FIII9–10 mimics, toa large extent, the biological activity of the full-length fibronectinmolecule. However, FIII9 is conformationally unstable, evenin the context of the FIII9–10 pair. Here we report theconstruction of a series of hybrid mouse–human FIII9–10pairs that confer varying degrees of conformational stabilityto FIII9. The conformational stability of the human FIII9 modulewas increased 2–3-fold by substitution of Leu1408 withPro. We demonstrate that the biological activity of this mutantis enhanced. The resulting FIII9–10 mutant has good solutionproperties and will provide a template into which further mutationscan be incorporated in order to probe the structure–functionrelationship of the cell binding module of fibronectin.     

Functional display of Rhizomucor miehei lipase on surface of Saccharomyces cerevisiae with higher activity and its practical properties

BACKGROUND: A display system, which can translate DNA to functional peptides or proteins, is used as a new protein expression system. In this system, peptides or proteins are displayed on the cell surface as a fusion form with some anchoring proteins. Yeast cells displaying lipases on their cell‐surface could be used as whole‐cell biocatalysts. This research focuses on the functional display of Rhizomucor miehei lipase (RML) on the surface of Saccharomyces cerevisiae with higher activity. RESULTS: The lipases (RML) from R.miehei 3.4960 were of active form. The RML‐α‐agglutinin fusion proteins produced were not secreted into the culture media and were mostly immobilized on the yeast cells. Cell surface displayed lipase showed the highest activity at 45 °C and pH 8.0. CONCLUSION: The gene encoding RML from R.miehei 3.4960 can be functionally expressed on the cell surface of S. cerevisiae MT8‐1 using a glycosylphosphatidylinositol (GPI) anchor with higher activity. Copyright © 2007 Society of Chemical Industry     

铜绿假单胞菌外毒素A基因突变体的构建及其在大肠埃希菌中的表达

目的将铜绿假单胞菌外毒素A(Pseudomonas aeruginosa exotoxin A,PEA)基因突变为无毒性的ntPE基因,并在大肠埃希菌中进行表达。方法利用Primer Premier 5.0软件设计PEA基因引物及PEA基因第553位氨基酸缺失的突变引物,以铜绿假单胞菌(Pseudomonas aeruginosa,P.aeruginosa)基因组DNA为模板,PCR扩增PEA基因,插入pGEM-T载体中,构建重组克隆质粒pGEM-PEA,以其为模板,利用突变引物,扩增ntPE基因(无毒性PEA),插入pET-30a载体中,构建原核表达质粒pET-ntPE,转化大肠埃希菌BL21(DE3),IPTG诱导表达,表达产物经SDS-PAGE及Western blot分析。结果测序结果证明已成功获得突变基因;原核表达质粒经双酶切鉴定证明构建正确;表达的重组蛋白相对分子质量约69 000,主要以包涵体形式存在,表达量占菌体总蛋白的30%,可被小鼠抗PEA单克隆抗体特异性识别,具有较好的抗原性。结论已成功获得无毒性的PEA突变基因,为构建以PEA为蛋白佐剂的融合蛋白疫苗奠定了基础。