CO2 fixation by [WIVO(S2C2(CN)2)2]2?: functional model for the tungsten-formate dehydrogenase of Clostridium thermoaceticum

(NEt₄)₂[W^IVO(S₂C₂(CN)₂)₂] (1), isolated by reaction of Na₂ WO₄, Na₂S₂C₂(CN)₂ (Na₂mnt) in acidified (pH5.5) aqueous medium in the presence of excess of sodium dithionite and NEt₄Br, reduces CO₂/HCO ₃ ⁻ (pH 7.5) to yield HCOO⁻ and (NEt₄)₂[W^VIO₂(S₂C₂(CN)₂)₂] (2) mimicking tungsten-formate dehydrogenase (W-FDH) activity. (1) reacts with Na₂MoO₄ in acidic medium to produce [Mo^IvO(S₂C₂(CN)₂)₂]²⁻ implicating the displacement of tungsten by molybdenum from the cofactor complex in W-FDH.     

New insights from X-ray photoelectron spectroscopy into the chemistry of covalent enzyme immobilization, with glutamate dehydrogenase (GDH) on silicon dioxide as an example

A three-step process for immobilization of glutamate dehydrogenase (GDH) on the surface of silicon dioxide has been studied by X-ray photoelectron spectroscopy (XPS). The enzyme layer was deposited on the silicon dioxide surface after first exposing the surface to 3-aminopropyltriethoxysilane (3-APTS) and reacting the silylated surface with glutaraldehyde (GA). Fine XPS analysis, performed after each step of the chemical procedure, revealed unknown details of the step-by-step construction of the enzyme layer under different experimental conditions.     

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.     

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.     

CTAB-己醇-辛烷体系分离纯化醇脱氢酶的反萃研究

本文报道了用反胶束体系萃取醇脱氢酶(ADH)的研究结果。在此萃取体系中,以十六烷基三甲基溴化铵(CTAB)作为表面活性剂,辛烷和己醇作为溶剂和助溶剂。考察了表面活性剂及助溶剂浓度,水相pH值对ADH萃取的影响。详细讨论了离子强度、异丙醇浓度和振荡时间对ADH反萃的影响。确定了最佳反向萃取条件。     

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.