Stabilization of Escherichia coli isopropylmalate dehydrogenase by single amino acid substitution

To determine the key position for the unusual stability of isopropylmalate dehydrogenase from extreme thermophiles (Thermus thermophilus and T. aquaticus), sequence comparisons were carried out. As a result, a motif which is characteristic to the thermophilic dehydrogenases was found between two highly conserved stretches. The sequence motif was introduced into a mesophilic (Escherichia coli) isopropylmalate dehydrogenase, one by one. Contrary to our expectation, introduction of the whole motif led the mesophilic enzyme to be more unstable whereas substitution of only one amino acid residue in the motif thermostabilized the enzyme. From the 3D structure of the enzyme, a mechanism for the thermostabilization is speculated.      

Engineering the aggregation properties of dodecameric glutamine synthetase: a single amino acid substitution controls 'salting out'

Escherichia coli glutamine synthetase (GS) is a dodecamer ofidentical subunits which are arranged as two face-to-face hexamericrings. In the presence of 10% ammonium sulfate, wild type GSexhibits a pH-dependent ‘salting out’ with a pK_aof 4.51. Electron micrographs indicate that the pH-dependentaggregation corresponds to a highly specific self-assembly ofGS tubules, which result from stacking of individual dodecamers.This stacking of dodecamers is similar to the metal ion-inducedGS tubule formation previously described. Site-directed mutagenesisexperimentsindicate that the N-terminal helix of each subunit is involvedin the salting out reaction, as it is in the metal-induced stacking.A single substitution of alanine for His4 completely abolishesthe (NH₄₂SO₄-induced aggregation. However, the H4C mutant proteindoes nearly completely precipitate under the same salting outconditions. Mutations at other residues within the helix haveno effect on the stacking reaction. Differential catalyticactivityof unadenylylated GS versus adenylylated GS has been used todetermine whether wild type dodecamers ‘complement’the H4A mutant in the stacking reaction. The complementationexperiments indicate that His4 residues on bothsides of thedodecamer-dodecamer interfaces are not absolutely required forsalting out, although the wild type dodecamers clearly stackpreferentially with other wild type dodecamers. Approximately20% of the protein precipitated fromthe mixtures containingthe wild type GS and the H4A mutant is the mutant. The implicationsof these results for protein engineering are discussed.     

Directed evolution of Tk-subtilisin from a hyperthermophilic archaeon: identification of a single amino acid substitution responsible for low-temperature adaptation

Tk-subtilisin from the hyperthermophilic archaeon Thermococcus kodakaraensis is synthesized in a prepro-form (prepro-Tk-subtilisin), secreted in a pro-form (pro-Tk-subtilisin), and matured to an active form (mat-Tk-subtilisin*; a Ca(2+)-bound active form of matured domain of Tk-subtilisin) upon autoprocessing and degradation of the propeptide [Tk-propeptide; propeptide of Tk-subtilisin (Gly1-Leu69)]. Pro-Tk-subtilisin exhibited halo-forming activity only at 80 degrees C, but not at 70 and 60 degrees C, because Tk-propeptide is not effectively degraded by mat-Tk-subtilisin* and forms an inactive complex with mat-Tk-subtilisin* at <80 degrees C. Random mutagenesis in the entire prepro-Tk-subtilisin gene, followed by screening for mutant proteins with halo-forming activity at 70 and 60 degrees C, allowed us to identify single Gly56 --> Ser mutation in the propeptide region responsible for low-temperature adaptation of pro-Tk-subtilisin. SDS-PAGE analyses and mat-Tk-subtilisin* activity assay of pro-G56S-subtilisin indicated more rapid maturation than pro-Tk-subtilisin. The resultant active form was indistinguishable from mat-Tk-subtilisin* in activity and stability, indicating that Gly56 --> Ser mutation does not seriously affect the folding of the mature domain. However, this mutation greatly destabilized the propeptide, making it unstructured in an isolated form. As a result, Tk-propeptide with Gly56 --> Ser mutation (G56S-propeptide) was more susceptible to proteolytic degradation and less effectively inhibited mat-Tk-subtilisin* activity than Tk-propeptide. These results suggest that pro-G56S-subtilisin is more effectively matured than pro-Tk-subtilisin at lower temperatures, because autoprocessed G56S-propeptide is unstructured upon dissociation from mat-Tk-subtilisin* and is therefore effectively degraded by mat-Tk-subtilisin*.     

Decreasing the stability and changing the substrate specificity of the Bacillus stearothermophilus alcohol dehydrogenase by single amino acid replacements

The gene encoding the alcohol dehydrogenase (adh-hT) from the thermophilicbacterium Bacillus stearothermophilus LLD-R strain has been overexpressedin Escherichia coli and the corresponding recombinant protein purified tohomogeneity. Two putative structural determinants contributing to thehigher stability of ADH-hT had been identified by comparison with the lessthermostable ADH (ADH-T) from the less thermophilic B. stearothermophilusNCA 1503. In order to ascertain their role, mutations were designed toeliminate in ADH-hT a salt bridge at the N-terminus and a proline residuein the coenzyme binding domain replacing the amino acids located at thesame positions in ADH-T. Three mutants--Glu11Lys, Pro242Ala, andGlu11Lys/Pro242Ala-- were expressed at high level and the proteins purifiedand characterized. In general, the mutations had little effect on theactivity, indicating that they were not disruptive. The thermal resistancewas changed displaying quite additive effects.     

In silico identification of structure requirement for novel thiazole and oxazole derivatives as potent fructose 1,6-bisphosphatase inhibitors

Fructose 1,6-bisphosphatase (FBPase) has been identified as a drug discovery target for lowering glucose in type 2 diabetes mellitus. In this study, a large series of 105 FBPase inhibitors were studied using a combinational method by 3D-QSAR, molecular docking and molecular dynamics simulations for a further improvement in potency. The optimal 3D models exhibit high statistical significance of the results, especially for the CoMFA results with r(ncv) (2), q(2) values of 0.986, 0.514 for internal validation, and r(pred) (2), r(m) (2) statistics of 0.902, 0.828 statistics for external validation. Graphic representation of the results, as contoured 3D coefficient plots, also provides a clue to the reasonable modification of molecules. (1) Substituents with a proper length and size at the C5 position of the thiazole core are required to enhance the potency; (2) A small and electron-withdrawing group at the C2 position linked to the thiazole core is likely to help increase the FBPase inhibition; (3) Substituent groups as hydrogen bond acceptors at the C2 position of the furan ring are favored. In addition, the agreement between 3D-QSAR, molecular docking and molecular dynamics simulation proves the rationality of the developed models. These results, we hope, may be helpful in designing novel and potential FBPase inhibitors.     

A single amino acid substitution can restore the solubility of aggregated colicin A mutants in Escherichia coli

Mutants of colicin A have been prepared in which the three tryptophanresidues (Trp86, Trpl30 and Trpl40), localized in the C-terminaldomain of the soluble wild-type protein, have been substitutedby phenylalanine. The Trpl40Phe single mutation had the effectof decreasing the percentage of protein that is expressed asinsoluble aggregates. The created hydrophobic cavity decreasedthe stability of the protein during its folding, resulting inpartial aggregation in the cytoplasm of the Escherichia coli-producingcells. Aggregation was increased when Trpl40 was substitutedby Lys, Leu or Cys, or if the Trpl40 mutation was combined withthe Trp86Phe and/ or Trpl30Phe mutations. A single mutation,Lysll3Phe, however, was able to restore the solubility of theaggregated mutants in vivo. Detailed analysis of the 3-D structureof the C-terminal domain of colicin A suggests that fillingof the hydrophobic cavity is responsible for this effect.     

Single amino acid substitutions can further increase the stability of a thermophilic L-lactate dehydrogenase

Lactate dehydrogenases are of considerable interest as stereospecificcatalysts in the chemical preparation of enantiomerically pure-hydroxyacid synthons. For such applications in synthetic organicchemistry it would be desirable to have enzymes which tolerateelevated temperatures for prolonged reaction times, to increaseproductivity and to extend then applicability to poor substrates.Here, two examples are reported of significant thermostabilizations,induced by sitedirected mutagenesis, of an already thermostableprotein, the L-lactate dehydrogenase (EC 1.1.1.27 [EC] , 35 kDa permonomer subunit) from Bacillus stearothermophilus. Thermal inactivationof this enzyme is accompanied by irreversible unfolding of thenative protein structure. The replacement of Argl71 by Tyr stabilizesthe enzyme against thermal inactivation and unfolding. Thisstabilizing effect appears to be based on improved interactionsbetween the subunits in the core of the active dimeric or tetramericforms of the enzyme. The thermal stability of L-lactate dehydrogenasevariants with an active site Arg residue, either in the 171(wild-type) or in the 102 position, is further increased bysulfate ions. The two stabilizing effects are additive, as foundfor the Argl71Tyr/ Gln1O2Arg double mutant, for which the stabilityof the protein in 100 mM sulfate solution reaches that of L-lactatedehydrogenases from extreme thermophiles. All mutant proteinsretain significant catalytic activity, both in the presenceand absence of stnhilfoing salts, and are viable catalysts inpreparative scale reactions.     

Analysis of the effect of accumulation of amino acid replacements on activity of 3-isopropylmalate dehydrogenase from Thermus thermophilus

A newly selected cold-adapted mutant 3-isopropylmalate dehydrogenase(IPMDH) from a random mutant library was a double mutant containingthe mutations I11V and S92F that were found in cold-adaptedmutant IPMDHs previously isolated. To elucidate the effect ofeach mutation on enzymatic activity, I11V and six multiple mutantIPMDHs were constructed and analyzed. All of the multiple mutantIPMDHs were found to be improved in catalytic activity at moderatetemperatures by increasing the k_cat with a simultaneous increaseof K_m for the coenzyme NAD⁺. k_cat was improved by a decreasein the activation enthalpy, H. The multiple mutants did notshow large reduction in thermal stability, and one of them showedenhanced thermal stability. Mutation from I11 to V was revealedto have a stabilizing effect. Mutants showed increased thermalstability when the mutation I11V was combined. This indicatesthat it is possible to construct mutants with enhanced thermalstability by combining stabilizing mutation. No additivity wasobserved for the thermodynamic properties of catalytic reactionin the multiple mutant IPMDHs, implying that the structuralchanges induced by the mutations were interacting with eachother. This indicates that careful and detailed tuning is requiredfor enhancing activity in contrast to thermal stability.     

耐有机溶剂氨基酸脱氢酶基因挖掘与非天然氨基酸的非水相合成

可在非水相体系高效催化不对称还原反应制备手性化合物的氧化还原酶具有重要科学意义与工业应用前景。基于基因挖掘技术,获得了17个耐盐氨基酸脱氢酶的基因,并分析了其进化同源性和蛋白质稳定性热力学参数。选取来源于Natranaerobius thermophilus的苯丙氨酸脱氢酶（PheDH）,进行了基因合成和表达、分离和纯化,获得了耐盐氨基酸脱氢酶,并检测了其有机溶剂耐受性。结果表明,对于催化L-苯丙氨酸的氧化脱氨体系,反应的最适温度为60℃,最适pH为 12。在含有30%的二甲亚砜反应体系中,催化活性是水相体系的1.2倍。而对于催化还原胺化制备L-高苯丙氨酸的体系,最适温度为70℃,最适pH为8.5。在含30%的甲基叔丁基醚和二甲亚砜反应体系中,催化活性分别是原始活性的101.3%和99.2%。研究表明,该耐盐酶具有较好的耐热、耐有机溶剂等抗逆性能。     

Generation of acid catalytic activity in Si-VPI-5 by partial Si for P substitution during hydrothermal synthesis

Si-VPI-5 crystals containing Si atoms in Si(4Al) environments arc synthesized. The crystals contain occluded amine. It is protonated and neutralizes the residual negative lattice charges associated with the Si(4A1) sites. Calcined Si-VPI-5 is active in the isomerisation of decane and exhibits extra-large pore characteristics.     

Alteration of the amino acid substrate specificity of clostridial glutamate dehydrogenase by site-directed mutagenesis of an active-site lysine residue

Two residues, K89 and S380, thought to interact with the -carboxylgroup of the substrate L-glutamate, have been altered by site-directedmutagenesis of clostridial glutamate dehydrogenase (GDH). Thesingle mutants K89L and S380V and the combined double mutantK89L/S380V were constructed. All three mutants were satisfactorilyoverproduced in soluble form. However, only the K89L mutantwas retained by the dye column normally used in purifying thewild-type enzyme. All three mutant enzymes were purified tohomogeneity and tested for substrate specificity with 24 aminoacids. The single mutant S380V showed no detectable activity.The alternative single mutant K89L showed an activity towardsL-glutamate that was decreased nearly 2000-fold compared withwild-type enzyme, whereas the activities towards the monocarboxylicsubstrates -aminobutyrate and norvaline were increased 2- to3-fold. A similar level of activity was obtained with methionine(0.005 U/mg) and norleucine (0.012 U/mg), neither of which giveany activity with the wild-type enzyme under the same conditions.The double mutant showed decreased activity with all substratescompared with the wild-type GDH. In view of its novel activities,the K89L mutant was investigated in greater detail. A strictlylinear relationship between reaction velocity and substrateconcentration was observed up to 80 mM L-methionine and 200mM L-norleucine, implying very high K_m values. Values of k_cat/K_m,for L-methionine and L-norleucine were 6.7x10^–2 and 0.15s^–1M^–1, respectively. Measurements with dithiobisnitrobenzoicacid showed that the mutant enzymes all reacted with a stoichiometryof one -SH group per subunit and all showed protection by coenzyme,indicating essentially unimpaired coenzyme binding. With glutamateor 2-oxoglutarate as substrate the K_m values for the vestigialactivity in the mutant enzyme preparations were strikingly closeto the wild-type K_m values. Both for wild-type GDH and K89L,L-glutamate gave competitive product inhibition of 2-oxoglutaratereduction but did not inhibit the reduction of 2-oxocaproatecatalysed by K89L enzyme. This suggests that the low levelsof glutamate/2-oxoglutarate activity shown by the mutant enzymeare due to trace contamination. Since stringent precautionswere taken, it appears possible that this reflects the levelof reading error during overexpression of the mutant proteins.CD measurements indicate that the S380V mutant has an alteredconformation, whereas the K89L enzyme gave an identical CD spectrumto that of wild-type GDH; the spectrum of the double mutantwas similar, although somewhat altered in intensity. The resultsconfirm the key role of K89 in dicarboxylate recognition byGDH.     

Increased promiscuity of human galactokinase following alteration of a single amino acid residue distant from the active site

Galactokinase catalyses the site- and stereospecific phosphorylation of galactose at the expense of ATP. The specificity of bacterial galactokinase enzymes can be broadened by alteration of a tyrosine residue to a histidine. The effects of altering the equivalent residue in human galactokinase (Tyr379) were investigated by testing all 19 possible variants. All of these alterations, except Y379P, resulted in soluble protein on expression in Escherichia coli and all the soluble variants could catalyse the phosphorylation of galactose, except Y379A and Y379E. The variants Y379C, Y379K, Y379R, Y379S and Y379W were all able to catalyse the phosphorylation of a variety of monosaccharides, including ones that are not acted on by the wild-type enzyme. Novel substrates for these variant galactokinases included D-mannose and D-fructose. The latter monosaccharide is presumed to react in the pyranose configuration. Molecular modelling suggested that the alterations do not cause changes to the overall structure of the enzyme. However, alteration of Tyr379 increases the flexibility of the peptide backbone in regions surrounding the active site. Therefore, it is proposed that alteration of Tyr379 affects the substrate specificity by the propagation of changes in flexibility to the active site, permitting a broader range of compounds to be accommodated.     

德氏乳杆菌发酵生产乳酸工艺条件优化

本实验在选育高产菌株及优化培养基的基础上,通过探索德氏乳杆菌的发酵工艺提高其乳酸产量。用初始菌株在基础发酵培养基上进行乳酸（DL-乳酸）发酵,其中L-乳酸的产量为18.5 g/L,对葡萄糖的转化率为50.27%。对原始菌株进行了激光诱变,选育出一株高产菌株DBLB28,对其培养基进行了进一步优化。先用正交实验设计法选出了主要的影响因子,然后再利用响应面法对所选因子进行优化,最优的条件为：葡萄糖浓度68.6 g/L;牛肉膏浓度22.4 g/L;蛋白胨浓度3.2 g/L,从回归方程预计乳酸的最大产量为29.0 g/L。在以上优化条件下进行发酵实验,测得乳酸产量为29.4 g/L,与预计值吻合较好。     

Single amino acid substitution in the mouse IgG1 Fc region induces drastic enhancement of the affinity to protein A

The purification of monoclonal antibody sometimes requires alot of time and involves complicated steps because of the poorerability of mouse IgG to interact with protein A, or also withprotein G, than IgGs from other species such as those of humanand rabbit. To resolve this problem, we exchanged one or twoamino acid residues of mouse IgG Fc region with that of humanIgG. Three mutants (T252M, T254S and T252M–T254S) showedsignificant improvement in the affinity to protein A. The exchangeof the threonine 252 residue to methionine (T252M) was mostefficient. This result suggests that a direct and simple modificationallows the efficient purification of monoclonal antibody andof fusion protein containing mouse IgG Fc region.     

离子液体作为微波吸收剂用于促进亲核取代反应

微波辐射下,乙氧甲叉丙二酸亚异丙酯与芳胺、酚等亲核试剂在离子液体中能够快速、高效的发生亲核取代反应生成产物。与传统方法相比,反应时间极大地缩短,产物结构经核磁共振确证。     

Polymorphism and phase changes in the ferrite phase of cements induced by titanium substitution

The introduction of TiO₂ at 1–10% levels into the crystal structure of the ferrite phase, nominally Ca₂(Fe_2?xA?_2x)O₅, has been studied experimentally by X-ray and electron diffraction. Ti⁴⁺ substitutes in ferrite mainly in octahedral sites, replacing Fe³⁺. Charge compensation is maintained by adding extra oxygens. At low Ti contents, typically 1–9%, the oxygen distributions are random and the crystal structure extensively disordered in the b direction. However, at higher Ti contents, an ordered superstructure with a b axis periodicity of /t 18.6 Å develops. This phase represents a different stacking arrangement than in C₄AF and has the ideal composition Ca₅(Fe_4/t-xA?_x)TiO₁₃. The appearance of powder X-ray diffraction patterns are interpreted in terms of the crystallographic changes.     

Structural evidence of a ketimine as the product of an amino acid with an aldehyde. An intermediate in the racemization and transamination of amino acids

The aldimine and ketimine forms of two Schiff base complexes formed by the condensation of two isomeric imidazole carboxaldehydes with an amino acid are reported. Reaction of L¹, the Schiff base condensate of 5-methyl-4-imidazolecarboxaldehyde (5Me4Im) and valine, with copper(II) perchlorate results in the isolation of [Cu(L¹)(5Me4Im)(ClO₄)] while the analogous reaction of L², the Schiff base condensate of 1-methyl-2-imidazolecarboxaldehyde (1Me2Im) with alanine, and nickel(II) results in the isolation of [Ni(L²)₂]. L¹ exhibits the expected aldimine form of the amino acid derived Schiff base, 5Me4Im-CH = N-CH(R)CO₂⁻, while L² exhibits the tautomeric ketimine form, 1Me2Im-CH₂–N = C(R’)CO₂⁻. Structural data clearly support the two tautomeric forms. The ketimine form, observed in [Ni(L²)₂], has been proposed as an intermediate in the racemization and transamination of amino acids.     

A new approach for the production of 2,5-furandicarboxylic acid by in situ oxidation of 5-hydroxymethylfurfural starting from fructose

The production of 2,5-furandicarboxylic acid (FDCA) starting with fructose as substrate via acid-catalyzed formation and subsequent oxidation of 5-hydroxymethylfurfural (HMF) was investigated. It was shown that an effective separation of the oxidation catalyst from fructose in combination with extraction and derivatization of formed HMF in methyl isobutyl ketone (MIBK) leads to formation of FDCA as final product. Two systems were developed to realize the concept, one by phase separation with a membrane, the other by encapsulating the oxidation catalyst in silicone beads. This revised version was published online in June 2006 with corrections to the Cover Date.     

Thermal conductivity of a single carbon nanocoil measured by field-emission induced thermal radiation

The spectrum of thermal radiation induced by field emission from a single carbon nanocoil (CNC) is obtained, from which its thermal conductivity is investigated. It is found that the resistivity of the CNC shows an exponential decrease with increasing temperature. The spectrum calculated by the temperature distribution along the CNC coincides with that calculated by the single maximum temperature in the wavelength range below 1250 nm, but shows considerable differences in intensity and peak position for higher wavelengths. Based on this understanding, the thermal conductivity of the CNC is evaluated to be 38 W/m K by a one-dimensional thermal conduction model.