Increasing the thermostability of Flavobacterium meningosepticum glycerol kinase by changing Ser329 to Asp in the subunit interface region

The thermostability enhancement of Flavobacterium meningosepticumglycerol kinase (FGK) by random mutagenesis in the subunit interfaceregion was investigated. A single Escherichia coli transformant,which produced a more thermostable glycerol kinase than theparent enzyme, was obtained. The nucleotide sequence of thegene of the mutant enzyme (FGK2615) was determined, and thefour amino acid replacements were identified as Glu327 to Asp,Ser329 to Asp, Thr330 to Ala and Ser334 to Lys. Although theproperties of FGK2615 were fundamentally similar to those ofthe parent enzyme, the thermostability and K_m for ATP had changed.The thermostability of FGK2615 was apparently increased; thetemperature at which the enzyme activity is inactivated by 50%for a 30-min incubation of FGK2615 was determined to be 72.1°Cwhich was 3.1°C higher than that of the parent FGK. Fouradditional mutants each having a single amino acid replacement(Glu327 to Asp, Ser329 to Asp, Thr330 to Ala and Ser334 to Lys)were prepared and their thermostability and K_m for substrateswere evaluated. The effect of the substitution of Ser329 toAsp is discussed.     

The structural basis of Trp192 and the C-terminal region in trichosanthin for activity and conformational stability

Trichosanthin (TCS) is a type I ribosome-inactivating protein(RIP) possessing N-glycosidase activity. TCS has various pharmacologicalproperties, including immunomodulatory, anti-tumor and anti-HIVactivities. Up to seven C-terminal residues of TCS (TCS-C7)can be deleted resulting in lower antigenicity with minimaleffects on its activity. However, an additional problem is thatthe minimal effects on activity are higher than the reductionin antigenicity. In the present work, the crystal structureof TCS-C7 was determined. It shows the details of the C-terminalresidues of TCS-C7, and in particular the hydrogen bonds betweenP35 and L240, S196 and L240, and W192 and L239, which play animportant role in maintaining the structure of TCS-C7. Furtheranalysis shows that the hydrogen bonds related to Leu240 arekey in maintaining the relationship between N- and C-terminaldomains. The major role of the C-terminal tail appears to stabilizethe structure of TCS. The conformation between helix H7 at theN-terminal domain and the C-terminal tail at the C-terminaldomain is also revealed. Two mutants, TCS-W192F and TCS-C7-W192F,were prepared and crystal structures were determined. Thesevariants have greatly reduced ribosome-inactivating activitiescompared with TCS and TCS-C7, respectively, and TCS-W192F andTCS-C7-W192F have a similar stability in guanidine hydrochloridecompared with TCS-C7. This suggests that Trp192 can affect theribosome-inactivating activity of TCS. Received November 27, 2002; revised March 7, 2003; accepted March 23, 2003.     

The highly exposed loop region in mammalian purple acid phosphatase controls the catalytic activity

Recombinant human purple acid phosphatase (recHPAP) provides a convenient experimental system for assessing the relationship between molecular structure and enzymatic activity in mammalian purple acid phosphatases (PAPs). recHPAP is a monomeric protein with properties similar to those of uteroferrin (Uf) and other PAPs isolated as single polypeptide chains, but its properties differ significantly from those of bovine spleen PAP (BSPAP) and other PAPs isolated as proteolytically "clipped" forms. Incubation of recHPAP with trypsin results in proteolytic cleavage in an exposed region near the active site. The product is a tightly associated two-subunit protein whose collective spectroscopic and kinetics properties resemble those of BSPAP. These results demonstrate that the differences in spectroscopic and kinetics properties previously reported for mammalian PAPs are the result of proteolytic cleavage. Mass spectrometry shows that a three-residue segment, D-V-K, within the loop region is excised by trypsin. This finding suggests that important interactions between residues in the excised loop and one or more of the groups that participate in catalysis are lost or altered upon proteolytic cleavage. Analysis of available structural data indicates that the most important such interaction is that between Asp 146 in the exposed loop and active-site residues Asn 91 and His 92. Loss of this interaction should result in both an increase in the Lewis acidity of the Fe(II) ion and an increase in the nucleophilicity of the Fe(III)-bound hydroxide ion. Proteolytic cleavage thus constitutes a potential physiological mechanism for regulating the activity of PAP in vivo.     

Enzymatic Synthesis and Molecular Modelling Studies of Rhamnose Esters Using Lipase from Pseudomonas stutzeri

Rhamnolipids are becoming an important class of glycolipid biosurfactants. Herein, we describe for the first time the enzymatic synthesis of rhamnose fatty acid esters by the transesterification of rhamnose with fatty acid vinyl esters, using lipase from Pseudomonas stutzeri as a biocatalyst. The use of this lipase allows excellent catalytic activity in the synthesis of 4-O-acylrhamnose (99% conversion and full regioselectivity) after 3 h of reaction using tetrahydrofuran (THF) as the reaction media and an excess of vinyl laurate as the acyl donor. The role of reaction conditions, such as temperature, the substrates molar ratio, organic reaction medium and acyl donor chain-length, was studied. Optimum conditions were found using 35 °C, a molar ratio of 1:3 (rhamnose:acyldonor), solvents with a low logP value, and fatty acids with chain lengths from C4 to C18 as acyl donors. In hydrophilic solvents such as THF and acetone, conversions of up to 99–92% were achieved after 3 h of reaction. In a more sustainable solvent such as 2-methyl-THF (2-MeTHF), high conversions were also obtained (86%). Short and medium chain acyl donors (C4–C10) allowed maximum conversions after 3 h, and long chain acyl donors (C12–C18) required longer reactions (5 h) to get 99% conversions. Furthermore, scaled up reactions are feasible without losing catalytic action and regioselectivity. In order to explain enzyme regioselectivity and its ability to accommodate ester chains of different lengths, homology modelling, docking studies and molecular dynamic simulations were performed to explain the behaviour observed.     

Probing the stabilizing role of C-terminal residues in trimethylamine dehydrogenase

In trimethylamine dehydrogenase, a homodimeric iron-sulfur flavoprotein,the C-terminal 17 residues of each subunit (residues 713- 729) embraceresidues on the other subunit. The role of this unusual mode of interactionat the subunit interface was probed by isolating three mutant forms oftrimethylamine dehydrogenase in which the C- terminus of the enzyme wasdeleted by five residues [delta(725-729], 10 residues [delta(720-729)] and17 residues [delta(713-729)]. The solution properties and conformationalstates of the three mutant enzymes were investigated using optical,fluorescence and circular dichroism spectroscopies, ANS binding and a noveland conformationally sensitive hydrodynamic method. The data reveal thatsequential deletion of the C-terminus of trimethylamine dehydrogenase doesnot affect significantly dimer stability or the overall structuralintegrity of the enzyme. However, deletion of the C-terminus severelycompromises, but does not abolish, the ability of the enzyme to becomecovalently coupled with the redox cofactor FMN in the active site, locatedover 20 A from the C-terminus. Hydrodynamic studies reveal minorconformational changes in the deletion mutants that lead to a more compactenzyme structure. These conformational changes are probably transmitted tothe active site via altering the interaction of the C-terminus with thesecond helix in the beta/alpha barrel of trimethylamine dehydrogenase,leading to poor flavinylation during the folding of the enzyme and assemblywith FMN.     

The role of the flap residue, threonine 77, in the activation and catalytic activity of pepsin A

Flexible loops, often referred to as flaps, have been shownto play a role in catalytic mechanisms of different enzymes.Flaps at the active site regions have been observed in the crystalstructures of aspartic proteinases and their residues implicatedin the catalytic processes. This research investigated the roleof the flap residue, threonine 77, in the activation of pepsinogenand the catalytic mechanism of pepsin. Three mutants, T77S,T77V and T77G, were constructed. Differences in amino acid polarityand hydrogen bonding potential were shown to have an influenceon the activation and catalytic processes. T77S activated atthe same rate and had similar catalytic parameters as the wild-typepepsin. The activation rates of T77V and T77G were slower andtheir catalytic efficiencies lower than the wild-type. The resultsdemonstrated that the threonine 77 polar side chain played arole in a proteolysis. The contribution of the side chain tozymogen activation was associated with the proteolytic cleavageof the prosegment. It was postulated that the hydroxyl groupat position 77 provided an essential hydrogen bond that contributedto proper substrate alignment and, indirectly, to a catalyticallyfavorable geometry of the transition state.     

On the role of acidity in catalytic oxidation

The role of the catalyst surface acid-base properties on the heterogeneously-catalyzed oxidation reaction mechanisms is discussed. Acid-base properties depend on the covalent/ionic character of the metal-oxygen bonds and are involved in some steps of the oxidation reactions, such as the activation of the C---H hydrocarbon bonds, the step associated with the evolution of alkoxide species and the desorption/overoxidation of the partial oxidation products. Thus they participate with the cation redox properties in determining the selective/unselective catalyst behavior.     

一种新催化体系的构建及其催化活性研究

用一种配体(5,5,7,12,12,14-六甲基-1,4,8,11-四氮杂环十四烷-N'-乙酸)和镧离子(Ⅲ)构建了一种新的催化体系,并用于催化对硝基苯酚磷酸二酯(BNPP)水解裂解。利用紫外-可见分光光度法研究了BNPP的催化水解的动力学。实验结果表明:这种新催化体系与其他相似的镧离子体系相比表现出更高的活性、水溶性和稳定性,在这一体系作用下,BNPP催化水解的速率比其自水解的速率提高了107倍左右。光谱分析表明,催化体系的活性物种是由镧离子和配体构成的大环配合物。     

Axial characterization of catalytic activity in close-coupled lightoff and underfloor catalytic converters

A exhaust system consisting of a close-coupled Pd technology 32 in³ lightoff converter and Pt/Rh technology 170 in³ underfloor converter was vehicle-aged for 56000 miles on a vehicle equipped with a 3.8 l engine. Following this aging, the converters were taken off the vehicle and cut into 1″ thick sections along their axis and characterized for lightoff and warmed-up activity using a laboratory reactor to simulate vehicle exhaust. Each section was also analyzed for the quantity of oil additive poisons (phosphorus and zinc) deposited. Following this initial characterization, the phosphorus and zinc deposits were removed, and the sections were characterized again for lightoff and warmed-up activity. This procedure was used to qualitatively determine the relative contribution of oil additive poisoning and thermal sintering to the total activity deterioration as a function of axial position in the catalyst monoliths.

Analysis of the lightoff converter as taken from the vehicle showed a dramatic axial gradient in the lean and stoichiometric lightoff and warmed-up (600°C) performance for HC, CO and NO_x, with most of the deterioration having taken place in the forward-most 1″ section of the converter, which was consistent with the gradient in the deposition of phosphorus (P) and zinc (Zn) in this converter. Comparison of these data sets with those obtained after removal of the P and Zn poisons indicates that most of the total deterioration of lean HC and CO activity can be attributed to P and Zn poisoning of the forwardmost 1″ section. When tested under stoichiometric conditions, most of the deterioration of HC activity is attributable to P and Zn poisoning, while most of the deterioration of CO and NO_x activity is attributable to thermal deterioration. A similar activity and poison deposition gradient was detected in the underfloor converter, but to a smaller extent.     

Engineering of the catalytic site of xylose isomerase to enhance bioconversion of a non-preferential substrate

Mutation in active site would either completely eliminate enzyme activity or may result in an active site with altered substrate-binding properties. The enzyme xylose isomerase (XI) is sterospecific for the α-pyranose and α-fructofuranose anomers and metal ions (M1 and M2) play a pivotal role in the catalytic action of this enzyme. Mutations were created at the M2 site of XI of Thermus thermophilus by replacing D254 and D256 with arginine. Mutants D254R and a double mutant (D254R/D256R) showed complete loss of activity while D256R showed an increase in the specificity on D-lyxose, L-arabinose and D-mannose which are non-preferential substrates for XI. Both wild type (WT) and D256R showed higher activity at pH 7.0 and 85°C with an increase in metal requirement. The catalytic efficiency Kcat/Km (S(-1) mM(-1)) of D256R for D-lyxose, L-arabinose and D-mannose were 0.17, 0.09 and 0.15 which are higher than WT XI of T.thermophilus. The altered catalytic activity for D256R could be explained by the possible role of arginine in catalytic reaction or the changes in a substrate orientation site. However, both the theories are only assumptions and have to be addressed with crystal study of D256R.     

梳状亲水性环氧基载体固定化Pseudomonas stutzeri LC2-8脂肪酶

以氯乙酰化聚苯乙烯微球(PS-acyl-Cl)为引发剂,亲水性丙烯酰胺(AM)和甲基丙烯酸缩水甘油酯(GMA)为单体,CuCl及联吡啶(Bipy)为催化体系,通过原子转移自由基聚合反应(ATRP)制备得到梳状亲水性环氧基柔性载体(PS-acyl-P(AM-co-GMA)),通过改变AM与GMA配比,使载体环氧基含量和亲水性得到控制.用该柔性载体固定Pseudomonas stutzeri LC2-8脂肪酶,优化了固定化条件,并对柔性固定化酶性质进行考察.结果显示,当n(AM)∶n(GMA)=20∶60,固定化时间为24 h,固定化温度为30℃,固定化pH为7.0时,固定化酶活力达到最高,为24.1U·g-1.固定化酶的最适pH为8.0,最适温度为30℃,其热稳定性比游离酶高,重复使用8次,剩余酶活力80％左右.以上表明,以ATRP法合成的载体PS-acyl-P(AM-co-GMA)可成功用于脂肪酶的固定化,有效提高脂肪酶的稳定性和实用性.     

The role of multiwalled carbon nanotubes in enhancing the catalytic activity of cobalt tetraaminophthalocyanine for oxidation of conjugated dyes

Multiwalled carbon nanotubes (MWCNTs) used to support a metallophthalocyanine catalyst (CoTAPc-MWCNTs) were prepared using covalent immobilization of cobalt tetraaminophthalocyanine (CoTAPc) on them, and characterized by X-ray photoelectron spectroscopy, attenuated total reflection Fourier transform infrared spectra and thermogravimetric analysis. The oxidative decoloration of rhodamine 6G (Rh6G) in the presence of CoTAPc-MWCNTs and H₂O₂ was investigated by examination of UV-Vis absorption spectra. The results showed that Rh6G was oxidized efficiently in the CoTAPc-MWCNTs/H₂O₂ system. The introduction of MWCNTs resulted in a marked enhanced catalytic activity that CoTAPc does not have. Electron paramagnetic resonance spin-trap experiments indicated that CoTAPc-MWCNTs have a novel non-radical pathway, which is different from common CoTAPc catalytic systems. Furthermore, the result of online electrochemical measurement in the CoTAPc-MWCNTs/H₂O₂ system suggested that MWCNTs might participate directly in the electron transfer process in the catalytic oxidation of this conjugated dye. In this catalytic system, MWCNTs provide strong adsorption to conjugated Rh6G due to their special sp²-hybridized surface, and are able to rapidly oxidize the conjugated dye by a special electron transfer pathway.     

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     

Isomerization ofn-butane on sulfated zirconia: Evidence for the dominant role of Lewis acidity on the catalytic activity

The catalytic activity of a ZrO₂/SO₄ catalyst in the isomerization ofn-butane at 423 K is reversibly suppressed by addition of CO. IR analysis of the adsorption of CO indicates that the only -coordination of CO onto coordinatively unsaturated surface Zr⁴⁺ cations occurs in the 300–473 K interval.     

Electrodeposition and catalytic activity of palladium powders

Palladium was deposited galvanostatically and potentiostatically from complex chloride solutions at various cds and potentials. The properties of the deposits were studied by a number of methods. The roughness measured with a mechanical profilometer strongly increased upon reaching the limiting cd in agreement with the general theory of the influence of mass transport control on the electrodeposition of metals.The particle size of the Pd-powders obtained above the limiting cd decreased steadily with increasing current density of deposition. In contrast to this, the BET-surface area as well as the catalytic activity (determined by measuring the acceleration of the rate of hydrogenation of cyclohexene) went through a maximum. The latter occurs in the region where the deposit consists of both the α and β H-Pd phases.The metal was deposited on a Pd-electrode as well as on a suspension of active carbon. The maximum catalytic activity was in both cases comparable to that of a non-electrolytic Pd-test catalyst.     

新型钼钒磷酸盐的合成及其催化性能

合成系列新型杂多酸盐Co(8-n)/2(Cpyr)nP2Mo16V2O62(n=1～8),用原子吸收光谱、傅里叶变换红外光谱、X射线衍射、热分析方法表征杂多化合物的结构,将其应用到乙苯氧化合成苯乙酮的反应中,并探讨其反应机理。结果表明,新型杂多酸盐Co(8-n)/2(Cpyr)nP2Mo16V2O62具有Dawson结构,过渡金属钴与活性中心钒之间存在协同作用,提高了催化剂的催化活性,其中Co2(Cpyr)4P2Mo16V2O62催化活性最高,乙苯的转化率达到83.7%,苯乙酮的收率达到74.8%。     

Selection of metalloenzymes by catalytic activity using phage display and catalytic elution

The metallo-beta-lactamase betaLII from Bacillus cereus 569/H/9 was displayed on the filamentous phage fd. The phage-bound enzyme fd-betaLII was shown to be active on benzylpenicillin as substrate; it could be inactivated by complexation of the essential zinc(II) ion with EDTA and reactivated by addition of a zinc(II) salt. A selection process was designed to extract active phage-bound enzymes from libraries of mutants in three steps: 1. inactivation of active phage-bound enzymes by metal ion complexation, 2. binding to substrate-coated magnetic beads, 3. release of phages capable of transforming the substrate into product upon zinc salt addition. The selection process was first successfully tested on model mixtures containing fd-betaLII plus either a dummy phage, a phage displaying an inactive mutant of the serine beta-lactamase TEM-1, or inactive and low-activity mutants of betaLII. The selection was then applied to extract active phage-bound enzymes from a library of mutants generated by mutagenic polymerase chain reaction (PCR). The activity of the library was shown to increase 60-fold after two rounds of selection. Eleven clones from the second round were randomly picked for sequencing and to characterize their activity and stability.     

The role of extra-framework chromium species for the catalytic activity of [Cr]APO-5-type materials

The properties of chromium-substituted aluminophosphate-5 ([Cr]APO-5) with and without extra-framework chromium generated during hydrothermal synthesis are compared with those of AlPO-5 and [Cr]APO-5 into which chromium species were partly or exclusively introduced by impregnation. The prepared materials were characterized by elemental analysis, XRD, UV–Vis spectroscopy and nitrogen adsorption. Extra-framework chromium species exhibit a slight activity for the acid-catalyzed gas-phase conversion of 2-propanol to propene. However, their major effect consists in a reduction of the specific pore volume and, thus, a decreased accessibility of acid sites in [Cr]APO-5 or AlPO-5. This pore volume reduction is more pronounced for materials with extra-framework chromium species from impregnation than from generation during synthesis.     

Site-directed mutagenesis of glyceraldehyde-3-phosphate dehydrogenase reveals the role of residue Ser148

A mutant of Bacillus stearothermophilus D-glyceraldehyde-3-phosphatedehydrogenase, Ser¹⁴⁸ – Ala, was produced byoligonucleotide-directedmutagenesis. The study of the catalytic properties of this mutanthas shown that this mutation significantly affects the Michaelisconstant of inorganic phosphate and to a lesser extent thatof 1,3-diphosphoglycerate and D-glyceraldehyde-3-phosphate.This result is consistent with model-building studies whichshow that, for the phosphorylation step of catalysis, inorganicphosphate must bind to the anion recognition site designatedP_i with the C(3) phosphate of the acyl-enzyme intermediate inthe alternative anion site P_s. Studies of the enantiomeric specificityusing D- and L-glyceraldehyde as substrates show that the hydroxylgroup of Ser¹⁴⁸, combined with the presence of the C(3) phosphateof the substrate, enhances stereospecificity as well as catalysis.However, the stereospecific effect cannot be a consequence ofthe direct interaction of Ser¹⁴⁸ with the C(2)-hydroxyl of thesubstrate. The changed K_m for glyceraldehyde-3-phosphate suggeststhat the initial step of hemithioacetal formation may take placewith its C(3) phosphate bound in the P_i site. This supportsthe molecular mechanism proposed by Moody (1984). Therefore,catalysis could be enhanced through interactions of the serinehydroxyl group not only with inorganic phosphate but also withthe C(3) phosphate of glyceraldehyde-3-phosphate.