Non‐natural Cofactor and Formate‐Driven Reductive Carboxylation of Pyruvate

A non‐natural cofactor and formate driven system for reductive carboxylation of pyruvate is presented. A formate dehydrogenase (FDH) mutant, FDH*, that favors a non‐natural redox cofactor, nicotinamide cytosine dinucleotide (NCD), for generation of a dedicated reducing equivalent at the expense of formate were acquired. By coupling FDH* and NCD‐dependent malic enzyme (ME*), the successful utilization of formate is demonstrated as both CO₂ source and electron donor for reductive carboxylation of pyruvate with a perfect stoichiometry between formate and malate. When ¹³C‐isotope‐labeled formate was used in in vitro trials, up to 53 % of malate had labeled carbon atom. Upon expression of FDH* and ME* in the model host E. coli, the engineered strain produced more malate in the presence of formate and NCD. This work provides an alternative and atom‐economic strategy for CO₂ fixation where formate is used in lieu of CO₂ and offers dedicated reducing power.     

Creation of bioorthogonal redox systems depending on nicotinamide flucytosine dinucleotide

Many enzymes catalyzing biological redox chemistry depend on the omnipresent cofactor, nicotinamide adenine dinucleotide (NAD). NAD is also involved in various nonredox processes. It remains challenging to disconnect one particular NAD-dependent reaction from all others. Here we present a bioorthogonal system that catalyzes the oxidative decarboxylation of l-malate with a dedicated abiotic cofactor, nicotinamide flucytosine dinucleotide (NFCD). By screening the multisite saturated mutagenesis libraries of the NAD-dependent malic enzyme (ME), we identified the mutant ME-L310R/Q401C, which showed excellent activity with NFCD, yet marginal activity with NAD. We found that another synthetic cofactor, nicotinamide cytosine dinucleotide (NCD), also displayed similar activity with the ME mutants. Inspired by these observations, we mutated d-lactate dehydrogenase (DLDH) and malate dehydrogenase (MDH) to DLDH-V152R and MDH-L6R, respectively, and both mutants showed fully active with NFCD. When coupled with DLDH-V152R, ME-L310R/Q401C required only a catalytic amount of NFCD to convert l-malate. Our results opened the window to engineer bioorthogonal redox systems for a wide variety of applications in systems biology and synthetic biology.     

Non-natural Cofactor and Formate-Driven Reductive Carboxylation of Pyruvate

A non-natural cofactor and formate driven system for reductive carboxylation of pyruvate is presented. A formate dehydrogenase (FDH) mutant, FDH*, that favors a non-natural redox cofactor, nicotinamide cytosine dinucleotide (NCD), for generation of a dedicated reducing equivalent at the expense of formate were acquired. By coupling FDH* and NCD-dependent malic enzyme (ME*), the successful utilization of formate is demonstrated as both CO₂ source and electron donor for reductive carboxylation of pyruvate with a perfect stoichiometry between formate and malate. When ¹³C-isotope-labeled formate was used in in vitro trials, up to 53 % of malate had labeled carbon atom. Upon expression of FDH* and ME* in the model host E. coli, the engineered strain produced more malate in the presence of formate and NCD. This work provides an alternative and atom-economic strategy for CO₂ fixation where formate is used in lieu of CO₂ and offers dedicated reducing power.     

Optimizing a biocatalyst for improved NAD(P)H regeneration: directed evolution of phosphite dehydrogenase

Cofactor regeneration is an important solution to the problem of implementing complex cofactor requiring enzymatic reactions at the industrial scale. NAD(P)H-dependent oxidoreductases are highly valuable biocatalysts, but the high cost of the nicotinamide cofactors necessitates in situ cofactor regeneration for preparative applications. Here we report the use of directed evolution to enhance the industrially important properties of phosphite dehydrogenase for NAD(P)H regeneration. A two-tiered sorting method of selection and screening was used in conjunction with random and rational mutagenesis. Following six rounds of directed evolution, soluble expression in E. coli was increased more than 3-fold, while the turnover rate was increased about 2-fold, effectively lowering the cost of the enzyme by >6-fold. Large-scale production of the final mutant enzyme by fermentation resulted in approximately 6-times higher yield (Units/Liter) than the WT enzyme. The enhancements of PTDH were independent of expression vector and E. coli strain utilized. The advantage of the final mutant over the WT enzyme was demonstrated using the industrially relevant bioconversion of trimethylpyruvate to L-tert-leucine. The mutations discovered are discussed in the context of a three dimensional structural model and the resulting changes in kinetics and soluble expression. The engineered phosphite dehydrogenase has great potential for NAD(P)H regeneration in industrial biocatalysis.     

Iron-doping Accelerating NADH Oxidation over Carbon Nitride

As a state-of-the-art conjugated polymer photocatalyst, graphitic carbon nitride(abbreviated as g-C₃N₄)has shown great potential in photocatalytic cofactor(reduced form of nicotinamide adenine dinucleotide, NADH) regene-ration. Herein, Fe-doped g-C₃N₄ was engineered for photocatalytic NADH oxidation. The π-π interaction between the NADH molecule and the conjugated heptazine building block facilitates the adsorption of NADH onto the framework, as revealed by density functional theory(DFT) calculations. Furthermore, iron doping promoted the oxidation kinetics of NADH under blue LED illumination. The conversion ratio of NADH to its oxidized form could be up to 85.7% in 20 min, comparing with 59.4% for metal-free counterpart. Enzyme assay employing formate dehydrogenase(FDH) further verified the selectivity of the products, with 67.5%±2.6% of enzymatically active 1,4-NADH being regenerated following the oxidation process. Scavenger experiments suggest the dominant role of photo-induced electrons in the oxidation of NADH. This work could shed light on developing a novel cofactor regeneration route through the synergistic effect between the metal doping and noncovalent interaction based on the conjugated polymer.     

20 Beta-hydroxysteroid dehydrogenase of neonatal pig testis: cofactor requirement and stereospecificity of hydrogen transfer from nicotinamide adenine dinucleotide phosphate, reduced form

The cofactor requirement of purified 20 beta-hydroxysteroid dehydrogenase from cytosol fraction of neonatal pig testis, in the reduction of 17 alpha-hydroxyprogesterone was investigated. The enzyme required beta-nicotinamide adenine dinucleotide phosphate, reduced form (beta-NADPH) as the preferred cofactor, with an apparent Km value of 17 microM. Furthermore, alpha-nicotinamide adenine dinucleotide phosphate, reduced form (alpha-NADPH), beta-3'-NADPH and beta-nicotinamide adenine dinucleotide (beta-NADH) were also utilized as hydrogen donors in the reduction at relatively high concentration with apparent Km values of 85.2 microM, 179.2 microM and 1.00 mM, respectively. The optimum pH was 5.5 when beta-NADPH was used as the cofactor, while it was 6.0 when beta-NADH was used. The hydrogen transfer from the beta-NADPH to the product, 17 alpha,20 beta-dihydroxypregn-4-en-3-one catalyzed by 20 beta-hydroxysteroid dehydrogenase was stereospecific, and the 4-pro-S-hydrogen of the nicotinamide moiety was transferred to the product.     

酶法测定乳酸

利用乳酸在氧化型烟酰胺腺嘌呤二核苷酸（NAD）存在的条件下,由乳酸脱氢酶（L-LDH）催化生成丙酮酸和还原型烟酰胺腺嘌呤二核苷酸（NADH）。通过测定NADH吸光度的变化率可得出乳酸的酶促反应速度,并制得标准曲线。样品中的乳酸可由标准曲线求得。讨论了试剂用量、pH、温度和共存离子对测定的影响。     

Molecular orbital study on the active center of lactate dehydrogenase

In the active site of lactate dehydrogenase important roles are given to aminoacids His195 and Arg171. The coenzyme nicotinamide adenine dinucleotide (NAD) is required in oxidized form (NAD⁺) for the enzymatic oxidation of the substrate L-lactate. Molecular orbital methods CNDO /2, INDO , and EHT were applied to the study of the model of active center of lactate dehydrogenase. An energetically preferred arrangement of the models of His195, Arg171, NAD⁺, and lactate was found. Possible biochemical aspects of these arrangements are discussed.     

Synthesis of NAD analogs to develop bioorthogonal redox system

Three new nicotinamide adenine dinucleotide(NAD) analogs were synthesized,and their characteristics as cofactors for Escherichia coli malic enzyme(ME) and its double mutant ME L310R/Q401C were analyzed.Each pair of the NAD analog and the double mutant showed good orthogonality to the natural pair of NAD and ME in terms of catalyzing oxidative decarboxylation of L-malic acid.Results indicated that molecular interactions between redox enzyme and cofactor could be further explored to generate new bioorthogonal redox systems.     

酶催化动力学荧光法测定乙醇

利用醇脱氢酶(ADH)催化乙醇与氧化型烟酰胺腺嘌呤二核苷酸(NAD)反应的原理,通过测定还原型烟酰胺腺嘌呤二核苷酸(NADH)荧光强度的变化率得出其酶促反应速度,对应乙醇浓度而制得标准曲线,试样中乙醇含量由标准曲线求得,检出限为4.0×10-6mol/L。考察了试剂用量、pH、共存组分对测定的影响。该方法简便、快速、准确。     

A theoretical study of the catalytic mechanism of formate dehydrogenase

A theoretical study of the hydride transfer between formate anion and nicotinamide adenine dinucleotide (NAD(+)) catalyzed by the enzyme formate dehydrogenase (FDH) has been carried out by a combination of two hybrid quantum mechanics/molecular mechanics techniques: statistical simulation methods and internal energy minimizations. Free energy profiles, obtained for the reaction in the enzyme active site and in solution, allow obtaining a comparative analysis of the behavior of both condensed media. Moreover, calculations of the reaction in aqueous media can be used to probe the dramatic differences between reactants state in the enzyme active site and in solution. The results suggest that the enzyme compresses the substrate and the cofactor into a conformation close to the transition structure by means of favorable interactions with the amino acid residues of the active site, thus facilitating the relative orientation of donor and acceptor atoms to favor the hydride transfer. Moreover, a permanent field created by the protein reduces the work required to reach the transition state (TS) with a concomitant polarization of the cofactor that would favor the hydride transfer. In contrast, in water the TS is destabilized with respect to the reactant species because the polarity of the solute diminishes as the reaction proceeds, and consequently the reaction field, which is created as a response to the change in the solute polarity, is also decreased. Therefore protein structure is responsible of both effects; substrate preorganization and TS stabilization thus diminishing the activation barrier. Because of the electrostatic features of the catalyzed reaction, both media preferentially stabilize the ground-state, thus explaining the small rate constant enhancement of this enzyme, but FDH does so to a much lower extent than aqueous solution. Finally, a good agreement between experimental and theoretical kinetic isotope effects is found, thus giving some credit to our results.     

脱氢酶电化学生物传感器的研究进展

自然界中超过400种脱氢酶使用辅酶-烟酰胺腺嘌呤二核苷酸(NAD+)或烟酰胺腺嘌呤二核苷酸磷酸(NADP+)作为生物催化反应中氢和电子的传递体,因此烟酰胺型辅酶的电化学氧化对构筑此类脱氢酶电化学生物传感器具有重要的意义.本文介绍了还原型辅酶在人工电子媒介体存在下的电化学氧化,以及脱氢酶电化学生物传感器的设计和应用.     

中空纤维膜固定化甲酸脱氢酶催化CO_2合成甲酸

以紫外光表面接枝改性的聚乙烯(PE)中空纤维膜为载体,采用共价结合的方式固定化甲酸脱氢酶(FDH),考察了CO2通入方式、溶液pH值、缓冲液种类和还原型烟酰胺腺嘌呤二核苷酸(NADH)的浓度对酶催化CO2合成甲酸反应的影响.结果表明,与加压法相比,CO2鼓泡法更有利于甲酸的生成;磷酸盐缓冲液优于Tris-HCl和盐酸三乙醇胺缓冲液;体系pH值对反应的影响较大,固定化FDH的最佳pH值仍为6.0,但pH耐受性增强;随着辅酶NADH浓度的增加,反应初速度加快,收率下降;游离酶和固定化酶的最大酶活分别为0.246和0.138mmol/(L.h);固定化FDH在4℃贮存两周后活性仅下降4%,而游离酶活性下降50%.FDH催化膜重复利用10次后,活性没有明显降低.     

A modular system for regeneration of NAD cofactors using graphite particles modified with hydrogenase and diaphorase moieties

Pyrolytic graphite particles modified with hydrogenase and an NAD(+)/NADH cycling enzyme provide a modular heterogeneous catalyst system for regeneration of oxidised or reduced nicotinamide cofactors using H(2) and H(+) as electron source or sink. Particles can be tuned for cofactor supply under different conditions by appropriate choice of hydrogenase.     

Amperometric Ethanol Biosensors Based on Chitosan‐NAD+‐Alcohol Dehydrogenase Films

The reagentless and oxygen‐independent biosensors for ethanol were developed based on the covalent immobilization of alcohol dehydrogenase (ADH) and its cofactor nicotinamide adenine dinucleotide (NAD⁺) on chitosan (CHIT) chains. The CHIT‐NAD⁺‐ADH structures were adsorbed onto carbon nanotubes (CNT) in order to provide a signal transduction based on the recycling of redox states of NAD cofactor at CNT (detection limit, 8–30 µM ethanol; dynamic range up to 20 mM). The CHIT‐NAD⁺‐dehydrogenase/CNT hybrid material represents a general approach to the development of dehydrogenases‐based electrochemical biosensors. Interestingly, the CHIT‐NAD⁺ solutions preserved their enzymatic activity even after five years of storage at 4 °C.     

Indirect electrochemical reduction of nicotinamide coenzymes

Nicotinamide coenzymes nicotinamide adenine dinucleotide (NAD(+)) and nicotinamide adenine dinucleotide phosphate (NADP(+)) were electrochemically reduced to NADH and NADPH, respectively. As direct reduction of nicotinamide coenzymes leads to inactive by-products, an indirect method using (pentamethylcyclopentadienyl-2,2'-bipyridine aqua) rhodium (III) as the mediator, was applied. A phosphate buffer solution, pH 8, with 1-10 mM NAD(P)(+) and 2.5-200 microM mediator, was pumped through a glassy carbon packed bed cathode. Virtually all the NAD(P)(+) was reduced to NAD(P)H in the cell. No sign of mediator loss due to side-reactions was detected though the mediator molecules shuttled hundreds of times between the oxidised and the reduced form. Adsorption of mediator molecules on the surface of the carbon cathode was found to be important for the reduction process. Due to strong adsorption, only minute amounts of mediator were consumed.     

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.     

Development of a high-performance liquid chromatographic system with enzyme reactors for the determination of N-acetyl branched-chain amino acids

Immobilized enzymes were used as column reactors in a high-performance liquid chromatographic system for the specific detection of N-acetyl branched-chain amino acids (AcBCAs) such as N- acetyl- l -valine (AcVal), N- acetyl- l -leucine (AcLeu) and N- acetyl- l -isoleucine (AcIle). Aminoacylase and leucine dehydrogenase were immobilized onto poly(vinyl alcohol) beads. The AcBCAs were separated as three peaks on a Capcell C(1) SG120 column with 0.03M phosphate buffer (pH 8.0). Aminoacylase was capable of hydrolysing the AcBCAs to amino acids, which react with beta-nicotinamide adenine dinucleotide (NAD(+)) in the presence of leucine dehydrogenase. The reduced nicotinamide adenine dinucleotide (NADH) produced was monitored fluorimetrically. The calibration graphs were linear from 4 to 200muM for AcVal and AcLeu, and from 5 to 300muM for AcIle; detection limits for AcVal, AcLeu and AcIle were 2, 2 and 3muM, respectively. The immobilized aminoacylase reactor should be renewed every 5 days owing to a poor stability of aminoacylase.     

新型烟酰胺腺嘌呤二核苷酸(NAD)类似物的合成及其辅酶活性

尝试4种形成焦磷酸键的方法合成了5种结构新颖的新型烟酰胺腺嘌呤二核苷酸(NAD)类似物.初步考察了类似物的生物活性,发现苹果酸酶和醇脱氢酶以类似物3b和3d为辅酶时,活性只有以NAD为辅酶时的13%～30%;而以类似物3a,3c和3e为辅酶时,这些酶的活性均极低.     

Bioinspired Photocatalytic NADH Regeneration by Covalently Metalated Carbon Nitride for Enhanced CO2 Reduction

Natural photosynthesis is a highly unified biocatalytic system, which coupled cofactor (NAD(P)H) regeneration and enzymatic CO₂ reduction efficiently for solar energy conversion. Mimicking nature, a novel system with Rh complex covalently grafted onto NH₂-functionalized polymeric carbon nitride (NH₂-PCN) was constructed. The integrated connection of the light-harvesting and electron mediation modules as Rh_m3-N-PCN could promote the efficient NAD⁺ reduction to NADH. As a result, the integrated system exhibited a conversion of ∼66 % within 20 minutes. By further coupling in situ generated NADH with formate dehydrogenase (FDH), a photoenzymatic production of formic acid (HCOOH) from CO₂ was accomplished. Moreover, by immobilizing FDH onto a hydrophobic membrane, an enhanced HCOOH production of ∼5.0 mM can be obtained due to the concentrated CO₂ on the gas-liquid-solid three-phase interface. Our work herein provides an integrated strategy for coupling the anchored electron mediator with immobilized enzyme for enhanced artificial photosynthesis.