Structure-Guided Design of Formate Dehydrogenase for Regeneration of a Non-Natural Redox Cofactor

Formate dehydrogenase (FDH) has been widely used for the regeneration of the reduced nicotinamide adenine dinucleotide (NADH). To utilize nicotinamide cytosine dinucleotide (NCD) as a non-natural redox cofactor, it remains challenging as NCDH, the reduced form of NCD, has to be efficiently regenerated. Here we demonstrate successful engineering of FDH for NCDH regeneration. Guided by the structural information of FDH from Pseudomonas sp. 101 (pseFDH) and the NAD–pseFDH complex, semi-rational strategies were applied to design mutant libraries and screen for NCD-linked activity. The most active mutant reached a cofactor preference switch from NAD to NCD by 3700-fold. Homology modeling analysis showed that these mutants had reduced cofactor binding pockets and dedicated hydrophobic interactions for NCD. Efficient regeneration of NCDH was implemented by powering an NCD-dependent D -lactate dehydrogenase for stoichiometric and stereospecific reduction of pyruvate to D -lactate at the expense of formate.     

Grafted Azure A modified electrodes as disposable β-nicotinamide adenine dinucleotide sensors

We report the in situ generation of aryl diazonium cations of Azure A, a redox-active phenothiazine dye, by reaction between the corresponding aromatic aminophenyl group and sodium nitrite in 0.1 M HCl. The subsequent electrochemical reduction of these dye diazonium salts gives rise to conductive electrografted films onto screen-printed carbon (SPC) electrodes. The resulting Azure A films have a very stable and reversible electrochemical response and exhibit potent and persistent electrocatalytic behavior toward NADH oxidation. We have optimized the electrografting conditions in order to obtain SPC modified electrodes with high and stable electrocatalytic response. The kinetic of the reaction between the NADH and the redox active centers in the Azure A film has been characterized using cyclic voltammetry and single step chronoamperometry. The catalytic currents were proportional to the concentration of NADH giving rise to linear calibration plots up to a concentration of 0.5 mM with a detection limit of 0.57 ± 0.03 μM and a sensitivity of 9.48 A mol cm⁻² μM⁻¹. The precision of chronoamperometric determinations was found to be 2.3% for five replicate determinations of 3.95 μM NADH. The great stability of such modified electrodes makes them ideal for their application in the development of biosensing platforms based on dehydrogenases.     

Detection of cationic guest molecules by quenching of luminescence of a self-assembled host molecule consisting of terbium(III) and calix[4]arene-p-tetrasulfonates

By self-assembly in aqueous solution, calix- (CAS) and thiacalix[4]arene-p-tetrasulfonate (TCAS) formed luminescent complexes Tb^III·(CAS)₂ and Tb^III·TCAS, respectively, which were utilized as a host for cationic guests. Addition of 1-ethylpyridinium guest quenched luminescence of Tb^III·(CAS)₂ in accordance with the Stern-Volmer (SV) relation with a low detection limit (D.L.) of 5.94 × 10⁻⁸ M (S/N = 3, M ≡ mol dm⁻³). On the other hand, 1-ethylquinolinium quenched luminescence of Tb^III·TCAS most efficiently, affording a very low D.L. (6.71 × 10⁻¹⁰ M). The agreement of the SV coefficients obtained with luminescent intensity (K_SV,all = 6.74 × 10⁶ M⁻¹) and lifetime (K_SV,Tb = 6.50 × 10⁶ M⁻¹) implied that dynamic quenching of ⁵D₄ excited state of Tb^III was predominant in the quenching processes. The quenching rate was estimated to be k_q,Tb = 9.94 × 10⁹ M⁻¹ s⁻¹, which was as fast as diffusion-limited rate. Quenching of Tb^III·(CAS)₂ was also applied to detection of NAD⁺, with a D.L. of 2.78 × 10⁻⁷ M.     

Synthesis of phosphodiester-type nicotinamide adenine dinucleotide analogs

Fourteen phosphodiester-type β-nicotinamide adenine dinucleotide (NAD⁺) analogs were prepared starting from nicotinamide. The phosphodiester linkage was effectively assembled in 69-93% yields via condensation reaction between 2′,3′-di-O-acetyl nicotinamide mononucleotide and alcohols in the presence of 2,4,6-triisopropylbenzenesulfonyl chloride. The analog β-nicotinamide ribose-5-(2-phenylethyl) phosphate showed beneficial effects on cell growth of model microorganisms.     

Photosensitization and photoprotection properties of nicotinic acid derivatives

The photosensitizing action of nicotinic acid, nicotinamide, and nicotinehydroxymethylamide on the photooxidation of glycyltryptophan (Gly-Trp) in an aqueous solution using UV light (240–410 nm) was found. The photooxidation was monitored by measuring chemiluminescence (CL) resulting from the decay of one of the oxidation products, dioxethane. The photosensitizing action decreases in the following sequence: nicotinamide > nicotine-hydroxymethylamide > nicotinic acid. The addition of benzoquinone (0.01 mmol L⁻¹) results in a substantial decrease in the yield of sensitized CL, which indicates that the superoxide radical anion participates in the photooxidation. Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 5, pp. 942–945, May, 1997.     

Sample preparation workflow for the liquid chromatography tandem mass spectrometry based analysis of nicotinamide adenine dinucleotide phosphate cofactors in yeast†

The accurate quantification of the highly unstable intracellular cofactor nicotinamide adenine dinucleotide phosphate in its oxidized and reduced forms demands a thorough evaluation of the analytical workflow and dedicated methods reflecting their solution chemistry as well as the biological importance of their ratio. In this work, we present a workflow for the analysis of intracellular levels of oxidized and reduced nicotinamide adenine dinucleotide phosphate in the yeast Pichia pastoris, including hot aqueous extraction, chromatographic separation in reversed‐phase conditions employing a 100% wettable stationary phase, and subsequent tandem mass spectrometric analysis. A thorough evaluation and optimization of the sample preparation procedure resulted in excellent biological repeatabilities (on average <10%, N = 3) without employing an internal standardization approach. As a consequence, the methodology proved to be appropriate for the relative assessment of intracellular levels of oxidized and reduced nicotinamide adenine dinucleotide phosphate in different P. pastoris strains. The ratio of reduced versus oxidized nicotinamide adenine dinucleotide phosphate was significantly higher in an engineered strain overexpressing glucose‐6‐phosphate dehydrogenase than in the corresponding wildtype strain. Interestingly, a difference was also observed in the nicotinamide adenine dinucleotide phosphate pool size, which was significantly higher in the wildtype than in the modified strain.     

基于对烟酰胺腺嘌呤二核苷酸光致电化学响应的胆汁酸传感器

将硫堇电聚合在多壁碳纳米管修饰电极上,然后在其表面固定3α-羟类固醇脱氢酶制备了胆汁酸传感器。 由于该传感器具有光敏和电子受体功能的光电界面,同时偶合了光致电化学效应和酶促反应,能与3α-羟类固醇脱氢酶催化胆汁酸产生的电子供体NADH(烟酰胺腺嘌呤二核苷酸)产生光致电化学反应,通过测量反应产生的光电流实现了对胆汁酸的检测。 探讨了胆汁酸传感器对胆汁酸的光致电化学响应机理,研究了多壁碳纳米管、酶固定量和硫堇初始浓度对光电极性能的影响。 在优化了电解质溶液pH值、辅酶用量和偏压的测试条件下,该传感器对胆汁酸的测定范围为1.80~40.0 μmol/L,检出限为0.67 μmol/L。 应用该传感器对药品胆酸钠片和人尿中胆汁酸进行的检测结果显示,相对标准偏差小于5.64%,加标回收率为96%~104%。 该传感器的制备和对胆汁酸的检测不需要除氧,有经济、简便等优点。     

痕量还原性辅酶Ⅰ的抑制荧光法测定

基于烟酰胺腺嘌呤二核苷酸 (简称辅酶I ,NADH)对血红蛋白催化H2 O2 氧化L 酪氨酸的反应具有强烈的抑制作用 ,提出了一种新的测定NADH的荧光分析法。该方法灵敏 ,简单 ,其线性范围为 5 .0× 1 0 -8～2 .0× 1 0 -6mol/L ;检出限为 2 .0× 1 0 -8mol/L。进一步探讨了NADH对血红蛋白催化反应的抑制机理 ,其抑制类型属于竞争性抑制     

烟酰胺辅酶在聚甲苯胺蓝膜修饰的玻碳电极上的电催化氧化

用循环伏安法在玻碳电极上电沉积一层稳定的甲苯胺蓝聚合物膜 ,研究了这层膜在 0 .2mol/L磷酸缓冲溶液 (pH 6 .86 )中的电化学性质 ,并且考察了该膜修饰的玻碳电极对烟酰胺辅酶 (NADH)的电催化作用 ,用旋转圆盘电极测量了NADH在该修饰电极上的催化反应常数。实验发现 ,在该修饰电极上 ,NADH氧化峰电位比未修饰的玻碳电极负移了 4 5 0mV ,且其催化反应速率常数为 3.5× 10 3 L·mol-1·s-1,说明聚甲苯胺蓝膜对NADH有良好的电催化作用     

黄素腺嘌呤二核苷酸在热石墨柱电极上电化学行为及吸附溶出分析

研究了黄素腺嘌呤二核苷酸(FAD)在热石墨柱电极上的电化学行为.发现对吸附有FAD的电极加热同时进行循环伏安扫描,随电极温度升高,氧化还原峰电位负移,峰电流减小,峰形变差.采用方波伏安进行吸附溶出分析,如果只在吸附步骤对电极加热,室温溶出,则随电极温度升高,溶出峰电流显著增加.这主要归因于直接对电极加热引起的强制热对流,这种对流能提高传质,促进吸附,增强溶出响应.5 min预富集对电极加热到72 ℃,检出限可以达到1×10-8 mol/L(S/N=3),比室温27 ℃时降低了一个多数量级,相应灵敏度也明显提高.