Equilibrium structure in the presence of internal rotation: A case study of cis-methyl formate

The Born-Oppenheimer (BO) equilibrium molecular structure () of cis-methyl formate has been determined at the CCSD(T) level of electronic structure theory using Gaussian basis sets of at least quadruple-ζ quality and a core correlation correction. The quadratic, cubic and semi-diagonal quartic force field in normal coordinates has also been computed at the MP2 level employing a basis set of triple-ζ quality. A semi-experimental equilibrium structure () has been derived from experimental ground-state rotational constants and the lowest-order rovibrational interaction parameters calculated from the ab initio cubic force field. To determine structures, it is important to start from accurate ground-state rotational constants. Different spectroscopic methods, applicable in the presence of internal rotation and used in the literature to obtain “unperturbed” rotational constants from the analysis and fitting of the spectrum, are reviewed and compared. They are shown to be compatible though their precision may be different. The and structures are in good agreement showing that, in the particular case of cis-methyl formate, the methyl torsion can still be treated as a small-amplitude vibration. The best equilibrium structure obtained for cis-methyl formate is: r(C_m-O) = 1.434 Å, r(O-C_c) = 1.335 Å, r(C_m-H_s) = 1.083 Å, r(C_m-H_a) = 1.087 Å, r(C_c-H) = 1.093 Å, r(CO) = 1.201 Å, ∠(COC) = 114.4°, ∠(CCH_s) = 105.6°, ∠(CCH_a) = 110.2°, ∠(OCH) = 109.6°, ∠(OCO) = 125.5°, and τ(H_aCOC) = 60.3°. The accuracy is believed to be about 0.001 Å for the bond lengths and 0.1° for the angles.     

SiO2负载Au-Pd双金属纳米颗粒催化甲醇选择性氧化合成甲酸甲酯

甲醇选择氧化制备甲酸甲酯（MF）是延伸甲醇产业链、开发高附加值下游产品的有效途径之一,负载型Au及Pd催化剂在这一反应中表现出优异的低温催化性能。为探索实用、高效和易再生的甲醇选择氧化催化剂,同时揭示双金属颗粒中Au和Pd的协同效应及甲醇氧化反应机理,本研究制备了一系列二氧化硅负载的Au-Pd催化剂（Au-Pd/SiO₂）,详细研究了其对甲醇选择氧化制甲酸甲酯的催化性能。结果表明,Au和Pd总负载量为0.6%、且Au/Pd质量比为2时,所制备的Au₂-Pd₁/SiO₂催化剂表现出优异的甲醇氧化催化性能;在130℃下,甲醇转化率达到57.0%,MF选择性为72.7%。多种表征结果显示,Au-Pd双金属纳米颗粒粒径为2-4 nm,高度分散于SiO₂载体表面,倾向于生成孪晶结构并暴露（111）晶面,这些因素是Au-Pd/SiO₂具有优异催化性能的主要原因。通过DRIFTS表征研究,提出了一个可能的MF生成机理：即甲醇首先与处于Au-Pd纳米粒子界面的表面氧作用,生成化学吸附的甲氧基;随后,甲氧基经去质子作用生成吸附的甲醛物种,后者与相邻的甲氧基物种亲核反应,并经β-H消除后得到目标产物MF。     

Structural insights of a cellobiose dehydrogenase enzyme from the basidiomycetes fungus Termitomyces clypeatus

Filamentous fungi secrete various oxidative enzymes to degrade the glycosidic bonds of polysaccharides. Cellobiose dehydrogenase (CDH) (E.C.1.1.99.18) is one of the important lignocellulose degrading enzymes produced by various filamentous fungi. It contains two stereo specific ligand binding domains, cytochrome and dehydrogenase - one for heme and the other for flavin adenine dinucleotide (FAD) respectively. The enzyme is of commercial importance for its use in amperometric biosensor, biofuel production, lactose determination in food, bioremediation etc. Termitomyces clypeatus, an edible fungus belonging to the basidiomycetes group, is a good producer of CDH. In this paper we have analyzed the structural properties of this enzyme from T. clypeatus and identified a distinct carbohydrate binding module (CBM) which is not present in most fungi belonging to the basidiomycetes group. In addition, the dehydrogenase domain of T. clypeatus CDH exhibited the absence of cellulose binding residues which is in contrast to the dehydrogenase domains of CDH of other basidiomycetes. Sequence analysis of cytochrome domain showed that the important residues of this domain were conserved like in other fungal CDHs. Phylogenetic tree, constructed using basidiomycetes and ascomycetes CDH sequences, has shown that very surprisingly the CDH from T. clypeatus, which is classified as a basidiomycetes fungus, is clustered with the ascomycetes group. A homology model of this protein has been constructed using the CDH enzyme of ascomycetes fungus Myricoccum thermophilum as a template since it has been found to be the best match sequence with T. clypeatus CDH. We also have modelled the protein with its substrate, cellobiose, which has helped us to identify the substrate interacting residues (L354, P606, T629, R631, Y649, N732, H733 and N781) localized within its dehydrogenase domain. Our computational investigation revealed for the first time the presence of all three domains - cytochrome, dehydrogenase and CBM - in the CDH of T. clypeatus, a basidiomycetes fungus. In addition to discovering the unique structural attributes of this enzyme from T. clypeatus, our study also discusses the possible phylogenetic status of this fungus.     

Exploring the influence of conserved lysine69 on the catalytic activity of the helicobacter pylori shikimate dehydrogenase: A combined QM/MM and MD simulations

Shikimate dehydrogenase (SDH) catalyzes the reversible, NADPH-dependent reduction of 3-dehydroshikimate to shikimate, involved in the shikimate pathway. This pathway has emerged as an important target for the development of antimicrobial agent. Structural and functional analyses suggest that the conserved Lys69 plays an important role in the catalytic activity of Helicobacter pylori (H. pylori) SDH. However, the detailed mechanism how mutation of Lys69 affects the catalytic activity of H. pylori SDH remains unclear. Here, two-layered ONIOM-based quantum mechanics/molecular mechanics (QM/MM) calculation and molecular dynamics (MD) simulations were performed to explore the role of Lys69 in the H. pylori SDH. Our results showed that in addition to act as a catalytic base, the conserved Lys69 plays an additional, important role in the maintenance of the substrate shikimate in the active site, facilitating the catalytic reaction between the cofactor NADP⁺ and shikimate. Mutation of Lys69 triggers the movement of shikimate away from the active site of SDH, thereby disrupting the catalytic activity. This result can advance our understanding the catalytic mechanism of SDH family, which may benefit of the rational design of SDH inhibitors.     

富晶格位错的多孔铋纳米花高效电还原二氧化碳制甲酸盐※

二氧化碳转化已成为现今世界研究的热点. 本工作采用原位电化学转化的策略, 将简单溶剂热法合成的层状甲酸氧铋纳米花(BiOCOOH NFs)还原为带有大量晶格位错的多孔铋纳米花(p-Bi NFs). 研究结果表明, p-Bi NFs电催化二氧化碳转化为甲酸盐具有较小的过电位(436 mV). 在–1.8 V(相对饱和甘汞电极, vs. SCE)时, 甲酸盐的分电流密度(j_formate)高达24.4 mA•cm^-2, 法拉第效率(FE_formate)为96.7%, 且在超过500 mV的宽电位窗口内FE_formate超过90%, 并具有很好的稳定性. 该催化剂的高催化性能可归因于前驱体晶格坍塌和重构而形成特殊的多孔粗糙的微纳多级结构, 其表面富含晶格位错和缺陷等高本征活性位, 且具有较强的电子传递能力. 本研究为设计合成高性能的电催化二氧化碳还原产甲酸催化剂提供了新的思路.     

Reagentless amperometric formaldehyde-selective biosensors based on the recombinant yeast formaldehyde dehydrogenase

Novel formaldehyde-selective amperometric biosensors were developed based on NAD(+)- and glutathione-dependent formaldehyde dehydrogenase isolated from a gene-engineered strain of the methylotrophic yeast Hansenula polymorpha. Electron transfer between the immobilized enzyme and a platinized graphite electrode was established using a number of different low-molecular free-diffusing redox mediators or positively charged cathodic electrodeposition paints modified with Os-bis-N,N-(2,2'-bipyridil)-chloride ([Os(bpy)(2)Cl]) complexes. Among five tested Os-containing redox polymers of different chemical structure and properties, complexes of osmium-modified poly(4-vinylpyridine) with molecular mass of about 60 kDa containing diaminopropyl groups were selected. The positively charged cathodic paint exhibited the best electron-transfer characteristics. Moreover, the polymer layers simultaneously served as a matrix for keeping the negatively charged low-molecular cofactors, glutathione and NAD(+), in the bioactive layer. Additionally, covering the enzyme/polymer layer with a negatively charged Nafion membrane significantly decreased cofactors leakage and simultaneously enhanced the sensor' stability. The developed sensors revealed a high selectivity to formaldehyde (FA) and a low cross-sensitivity to other substances (such as, e.g. butyraldehyde, propionaldehyde, acetaldehyde, methylglyoxal). The maximum current value was 34.2+/-0.72 microA/mm(2) (3.05 mm diameter electrode) and the apparent Michaelis-Menten constant (K(M)(app)) derived from the FA calibration curves was 120+/-5mM with a linear detection range for FA up to 20mM. The best observed sensitivity for reagentless sensor was 1.8 nA microM(-1) (358 Am(-2)M(-1)). The developed sensors had a good operational and storage stability. The laboratory prototype of the sensor was applied for FA testing in some real samples of pharmaceutical (formidron), disinfectant (descoton forte) and industrial product (formalin). A good correlation was revealed between the concentration values measured using the developed FdDH-based sensor, an enzymatic method and standard chemical methods of FA determination.     

Construction of L‐Lysine Sensor by Layer‐by‐Layer Adsorption of L‐Lysine 6‐Dehydrogenase and Ferrocene‐Labeled High Molecular Weight Coenzyme Derivative on Gold Electrode

A ferrocene‐labeled high molecular weight coenzyme derivative (PEI‐Fc‐NAD) and a thermostable NAD‐dependent L ‐lysine 6‐dehydrogenase (LysDH) from thermophile Geobacillus stearothermophilus were used to fabricate a reagentless L ‐lysine sensor. Both LysDH and PEI‐Fc‐NAD were immobilized on the surface of a gold electrode by consecutive layer‐by‐layer adsorption (LBL) technique. By the simple LBL method, the reagentless L ‐lysine sensor, with co‐immobilization of the mediator, coenzyme, and enzyme was obtained, which exhibited current response to L ‐lysine without the addition of native coenzyme to the analysis system. The amperometric response of the sensor was dependent on the applied potential, bilayer number of PEI‐Fc‐NAD/LysDH, and substrate concentration. A linear current response, proportional to L ‐lysine concentration in the range of 1–120 mM was observed. The response of the sensor to L ‐lysine was decreased by 30% from the original activity after one month storage.     

A Rigidoporus Vinctus Alcohol Dehydrogenase and Its Characterization

Alcohol dehydrogenases (ADHs; E.C. 1.1.1.1) are widely distributed enzymes found in many microorganisms. ADHs are oxidoreductases that catalyze the NAD(P)⁺‐dependent conversion of alcohols to aldehydes or ketones as well as the reverse reaction. The ADH cloned from Rigidoporus vinctus (RvADH) was 1035 bp that encodes a protein of 344 amino acid residues with calculated molecular mass of 38.39 kDa. This ADH is belonging to the medium‐chain family (medium‐chain dehydrogenase/reductase (MDR) and has the highly conserved GXXGXXG sequence found in the MDR family which found as the coenzyme‐binding pocket. To characterize the ADH protein, the coding region was subcloned into an expression vector pET‐20b(+) and transformed into E. coli Rosetta (DE3). The recombinant His6‐tagged ADH was overexpressed and purified by Ni²⁺‐nitrilotriacetic acid Sepharose. The purified enzyme showed one band on 12 % sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. The Michaelis constant (K_M) value of the recombinant enzyme for ethanol was 0.79 mM. In substrates specificity analysis showed that RvADH had great oxidative activity toward primary alcohols. However, the less activtiy toward secondary alcohols and alcohol derivatives were compared with ethanol. Regarding the reductase activity showed low or even no activity to aldehydes and ketone.