Mutation of Tyr-218 to Phe in <Emphasis Type="Italic">Thermoanaerobacter ethanolicus</Emphasis> Secondary Alcohol Dehydrogenase: Effects on Bioelectronic Interface Performance

Bioelectronic interfaces that facilitate electron transfer between the electrode and a dehydrogenase enzyme have potential applications in biosensors, biocatalytic reactors, and biological fuel cells. The secondary alcohol dehydrogenase (2° ADH) from Thermoanaerobacter ethanolicus is especially well suited for the development of such bioelectronic interfaces because of its thermostability and facile production and purification. However, the natural cofactor for the enzyme, β-nicotinamide adenine dinucleotide phosphate (NADP⁺), is more expensive and less stable than β-nicotinamide adenine dinucleotide (NAD⁺). PCR-based, site-directed mutagenesis was performed on 2° ADH in an attempt to adjust the cofactor specificity toward NAD⁺ by mutating Tyr²¹⁸ to Phe (Y218F 2° ADH). This mutation increased the K _m(app) for NADP⁺ 200-fold while decreasing the K _m(app) for NAD⁺ 2.5-fold. The mutant enzyme was incorporated into a bioelectronic interface that established electrical communication between the enzyme, the NAD⁺, the electron mediator toluidine blue O (TBO), and a gold electrode. Cyclic voltammetry, impedance spectroscopy, gas chromatography, mass spectrometry, constant potential amperometry, and chronoamperometry were used to characterize the mutant and wild-type enzyme incorporated in the bioelectronic interface. The Y218F 2° ADH exhibited a fourfold increase in the turnover ratio compared to the wild type in the presence of NAD⁺. The electrochemical and kinetic measurements support the prediction that the Rossmann fold of the enzyme binds to the phosphate moiety of the cofactor. During the 45 min of continuous operation, NAD⁺ was electrically recycled 6.7 × 10⁴ times, suggesting that the Y218F 2° ADH-modified bioelectronic interface is stable.     

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.     

20 beta-hydroxysteroid dehydrogenase of neonatal pig testis: reverse catalytic (oxidation) reaction

Neonatal pig testicular 20 beta-hydroxysteroid dehydrogenase (20 beta-HSD) catalyzed the oxidation of 20 beta-hydroxysteroids, 17 alpha,20 beta-dihydroxypregn-4-en-3-one and 20 beta-hydroxypregn-4-en-3-one in the presence of beta-nicotinamide adenine dinucleotide phosphate (beta-NADP+). The behavior of 20 beta-HSD activity toward the substrate of 17 alpha,20 beta-dihydroxypregn-4-en-3-one differed from the catalytic reaction for 20 beta-hydroxypregn-4-en-3-one. The enzyme could catalyze not only 20 beta-hydroxysteroids but also 20 alpha-hydroxy-5-ene steroids, 20 alpha-hydroxypregn-5-en-3 beta-ol and 17 alpha,20 alpha-hydroxypregn-5-en-3 beta-ol with 22.1 and 8.7% of activity relative to 20 beta-hydroxypregn-4-en-3-one, respectively. The enzyme preferentially required beta-NADP+, and also utilized beta-nicotinamide adenine dinucleotide beta-NAD+ and beta-nicotinamide adenine dinucleotide 3'-phosphate (beta-3'-NADP+) nonspecifically as the cofactor. The optimum pH was observed at pH 7.5 with the substrate of 20 beta-hydroxypregn-4-en-3-one. The activation energies obtained from oxidation-reduction reactions of 20 beta-HSD for the substrate of 20 beta-hydroxypregn-4-en-3-one, progesterone and 17 alpha-hydroxyprogesterone were estimated at 13.8, 27.0 and 20.0 kcal/mol, respectively.     

Integrated, electrically contacted NAD(P)+-dependent enzyme-carbon nanotube electrodes for biosensors and biofuel cell applications

Integrated, electrically contacted beta-nicotinamide adenine dinucleotide- (NAD(+)) or beta-nicotinamide adenine dinucleotide phosphate- (NADP(+)) dependent enzyme electrodes were prepared on single-walled carbon nanotube (SWCNT) supports. The SWCNTs were functionalized with Nile Blue (1), and the cofactors NADP(+) and NAD(+) were linked to 1 through a phenyl boronic acid ligand. The affinity complexes of glucose dehydrogenase (GDH) with the NADP(+) cofactor or alcohol dehydrogenase (AlcDH) with the NAD(+) cofactor were crosslinked with glutaric dialdehyde and the biomolecule-functionalized SWCNT materials were deposited on glassy carbon electrodes. The integrated enzyme electrodes revealed bioelectrocatalytic activities, and they acted as amperometric electrodes for the analysis of glucose or ethanol. The bioelectrocatalytic response of the systems originated from the biocatalyzed oxidation of the respective substrates by the enzyme with the concomitant generation of NAD(P)H cofactors. The electrocatalytically mediated oxidation of NAD(P)H by 1 led to amperometric responses in the system. Similarly, an electrically contacted bilirubin oxidase (BOD)-SWCNT electrode was prepared by the deposition of BOD onto the SWCNTs and the subsequent crosslinking of the BOD units using glutaric dialdehyde. The BOD-SWCNT electrode revealed bioelectrocatalytic functions for the reduction of O(2) to H(2)O. The different electrically contacted SWCNT-based enzyme electrodes were used to construct biofuel cell elements. The electrically contacted GDH-SWCNT electrode was used as the anode for the oxidation of the glucose fuel in conjunction with the BOD-SWCNT electrode in the presence of O(2), which acted as an oxidizer in the system. The power output of the cell was 23 muW cm(-2). Similarly, the AlcDH-SWCNT electrode was used as the anode for the oxidation of ethanol, which was acting as the fuel, with the BOD-SWCNT electrode as the cathode for the reduction of O(2). The power output of the system was 48 microW cm(-2).     

Immobilisation of enzymes on poly(aniline)-poly(anion) composite films. Preparation of bioanodes for biofuel cell applications.

Immobilisation of enzymes is important for applications such as biosensors or biofuel cells. A poly(histidine) tag had been introduced on the C terminus of a lactate dehydrogenase enzyme. This mutant enzyme was then immobilised onto poly(aniline) (PANi)-poly(anion) composite films, PANi-poly(vinylsulfonate) (PVS) or PANi-poly(acrylate) (PAA). The NADH produced by the immobilised enzyme in the presence of beta-nicotinamide adenine dinucleotide (NAD(+)) and lactate is oxidised at the poly(aniline)-coated electrode at 0.05 to 0.1 V vs. saturated calomel electrode (SCE) at 35 degrees C.     

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.     

Analysis of NAD(P) and NAD(P)H cofactors by means of imprinted polymers associated with Au surfaces:: A surface plasmon resonance study

Crosslinked films consisting of the acrylamide-acrylamidophenylboronic acid copolymer that are imprinted with recognition sites for β-nicotinamide adenine dinucleotide (NAD⁺), β-nicotinamide adenine dinucleotide phosphate NADP⁺, and their reduced forms (NAD(P)H), are assembled on Au-coated glass supports. The binding of the oxidized cofactors NAD⁺ or NADP⁺ or the reduced cofactors NADH or NADPH to the respective imprinted sites results in the swelling of the polymer films through the uptake of water. Surface plasmon resonance (SPR) spectroscopy is employed to follow the binding of the different cofactors to the respective imprinted sites. The imprinted recognition sites reveal selectivity towards the association of the imprinted cofactors. The method enables the analysis of the NAD(P)⁺ and NAD(P)H cofactors in the concentration range of 1×10⁻⁶ to 1×10⁻³ M. The cofactor-imprinted films associated with the Au-coated glass supports act as active interfaces for the characterization of biocatalyzed transformations that involve the cofactor-dependent enzymes. This is exemplified with the characterization of the biocatalyzed oxidation of lactate to pyruvate in the presence of NAD⁺ and lactate dehydrogenase using the NADH-imprinted polymer film.     

Improved chemical syntheses of 1- and 5-deazariboflavin

The cofactor flavin adenine dinucleotide (FAD) is required for the catalytic activity of a large class of enzymes known as flavoenzymes. Because flavin cofactors participate in catalysis via a number of different mechanisms, isoalloxazine analogues are valuable for mechanistic studies. We report improved chemical syntheses for the preparation of the two key analogues, 5-deazariboflavin and 1-deazariboflavin.     

Control release of lactate dehydrogenase encapsulated in poly (vinyl alcohol) nanofibers via electrospinning

Sustained release of lactate dehydrogenase (LDH, EC 1.1.1.27) from electrospun poly (vinyl alcohol) (PVA) nanofibers was successfully achieved using the coaxial electrospinning technique. The presence of the encapsulated enzyme in the nanofibers was confirmed by infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS). Scanning electron microscopy (SEM) was used to evaluate the morphology and diameter of the nanofibers. The conversion of lactate to pyruvate by LDH coupling with the reduction of the cofactor nicotinamide adenine dinucleotide (NAD⁺) to dihydronicotinamide adenine dinucleotide (NADH) produces an increment in the ultraviolet absorption (UV) at 340 nm. This change in the UV absorbance was used to follow the release kinetic of LDH from the PVA nanofibers and also as a measure to evaluate the residual enzymatic catalytic function. Most of the encapsulated LDH enzyme was released in a sustained manner from the PVA nanofibers within a period of 1 month.     

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.     

Electrical communication between electrode and dehydrogenase by a ferrocene-labeled high molecular-weight cofactor derivative: application to a reagentless biosensor

A ferrocene-labeled high molecular weight cofactor derivative (PEI-Fc-NAD) was prepared by attaching both ferrocene and nicotinamide adenine dinucleotide (NAD⁺) to a water soluble polyelectrolyte, polyethylenimine (PEI). Approximately 9.8% and 2.9% of all the primary amino groups of PEI were coupled with ferrocene and the bioactive cofactor, respectively. The cyclic voltammograms of PEI-Fc-NAD exhibited a one-electron transfer process, and the difference between the anodic and cathodic peak potential was found to be 80 mV. The PEI-Fc-NAD was used together with NAD-dependent dehydrogenase to construct a reagentless biosensor. An NAD-dependent alcohol dehydrogenase (ADH) was selected as the model enzyme, and both ADH and PEI-Fc-NAD were retained onto the sensing area of gold electrodes by a dialysis membrane or immobilized by layer-by-layer adsorption method. In both cases, the modified electrodes showed current response to ethanol without the addition of native NAD⁺ to the system, which suggested that the electrical communication between ADH and the electrode was achieved through PEI-Fc-NAD. In summary, PEI-Fc-NAD provides a new way for immobilization of mediator and cofactor, and exhibits the potential as a platform for constructing reagentless NAD-dependent dehydrogenase biosensors.     

Kinetics of befunolol reductase from rabbit liver

The kinetic mechanism for the reduction of befunolol catalyzed by befunolol reductase from rabbit liver was investigated. From the initial velocity analysis, product inhibition and coenzyme binding studies, the reduction of befunolol was found to proceed through an ordered Bi Bi mechanism, in which beta-nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) binds to the enzyme firstly and NADP+ leaves lastly. NADPH bound to the free enzyme at a molar ratio of 1:1. Furthermore, the result of dead-end inhibition by Cibacron blue F3GA, a nucleotide analogue which binds to many enzymes, was consistent with the ordered Bi Bi mechanism for the enzyme.     

Two-dimensional infrared spectroscopy of azido-nicotinamide adenine dinucleotide in water

Mid-IR active analogs of enzyme cofactors have the potential to be important spectroscopic reporters of enzyme active site dynamics. Azido-nicotinamide adenine dinucleotide (NAD(+)), which has been recently synthesized in our laboratory, is a mid-IR active analog of NAD(+), a ubiquitous redox cofactor in biology. In this study, we measure the frequency-frequency time correlation function for the antisymmetric stretching vibration of the azido group of azido-NAD(+) in water. Our results are consistent with previous studies of pseudohalides in water. We conclude that azido-NAD(+) is sensitive to local environmental fluctuations, which, in water, are dominated by hydrogen-bond dynamics of the water molecules around the probe. Our results demonstrate the potential of azido-NAD(+) as a vibrational probe and illustrate the potential of substituted NAD(+)-analogs as reporters of local structural dynamics that could be used for studies of protein dynamics in NAD-dependent enzymes.     

Probing Biomolecular Interactions at Conductive and Semiconductive Surfaces by Impedance Spectroscopy: Routes to Impedimetric Immunosensors,DNA‐Sensors,and Enzyme Biosensors

Impedance spectroscopy is a rapidly developing electrochemical technique for the characterization of biomaterial‐functionalized electrodes and biocatalytic transformations at electrode surfaces, and specifically for the transduction of biosensing events at electrodes or field‐effect transistor devices. The immobilization of biomaterials, e.g., enzymes, antigens/antibodies or DNA on electrodes or semiconductor surfaces alters the capacitance and interfacial electron transfer resistance of the conductive or semiconductive electrodes. Impedance spectroscopy allows analysis of interfacial changes originating from biorecognition events at electrode surfaces. Kinetics and mechanisms of electron transfer processes corresponding to biocatalytic reactions occurring at modified electrodes can be also derived from Faradaic impedance spectroscopy. Different immunosensors that use impedance measurements for the transduction of antigen‐antibody complex formation on electronic transducers were developed. Similarly, DNA biosensors using impedance measurements as readout signals were developed. Amplified detection of the analyte DNA using Faradaic impedance spectroscopy was accomplished by the coupling of functionalized liposomes or by the association of biocatalytic conjugates to the sensing interface providing biocatalyzed precipitation of an insoluble product on the electrodes. The amplified detections of viral DNA and single‐base mismatches in DNA were accomplished by similar methods. The changes of interfacial features of gate surfaces of field‐effect transistors (FET) upon the formation of antigen‐antibody complexes or assembly of protein arrays were probed by impedance measurements and specifically by transconductance measurements. Impedance spectroscopy was also applied to characterize enzyme‐based biosensors. The reconstitution of apo‐enzymes on cofactor‐functionalized electrodes and the formation of cofactor‐enzyme affinity complexes on electrodes were probed by Faradaic impedance spectroscopy. Also biocatalyzed reactions occurring on electrode surfaces were analyzed by impedance spectroscopy. The theoretical background of the different methods and their practical applications in analytical procedures were outlined in this article.     

Coordinated and reversible reduction of enzymes involved in terminal oxidative metabolism in skeletal muscle mitochondria from a riboflavin-responsive, multiple acyl-CoA dehydrogenase deficiency patient

In this case report we studied alterations in mitochondrial proteins in a patient suffering from recurrent profound muscle weakness, associated with ethylmalonic-adipic aciduria, who had benefited from high dose of riboflavin treatment. Morphological and biochemical alterations included muscle lipid accumulation, low muscle carnitine content, reduction in fatty acid beta-oxidation and reduced activity of complexes I and II of the respiratory chain. Riboflavin therapy partially or totally reversed these symptoms and increased the level of muscle flavin adenine dinucleotide, suggesting that aberrant flavin cofactor metabolism accounted for the disease. Proteomic investigation of muscle mitochondria revealed decrease or absence of several flavoenzymes, enzymes related to flavin cofactor-dependent mitochondrial pathways and mitochondrial or mitochondria-associated calcium-binding proteins. All these deficiencies were completely rescued after riboflavin treatment. This study indicates for the first time a profound involvement of riboflavin/flavin cofactors in modulating the level of a number of functionally coordinated polypeptides involved in fatty acyl-CoA and amino acid metabolism, extending the number of enzymatic pathways altered in riboflavin-responsive multiple acyl-CoA dehydrogenase deficiency.     

Role of adenine in thymine-dimer repair by reduced flavin-adenine dinucleotide

We present a study of excited-state behavior of reduced flavin cofactors using femtosecond optical transient absorption spectroscopy. The reduced flavin cofactors studied were in two protonation states: flavin-adenine dinucleotide (FADH2 and FADH-) and flavin-mononucleotide (FMNH2 and FMNH-). We find that FMNH- exhibits multiexponential decay dynamics due to the presence of two bent conformers of the isoalloxazine ring. FMNH2 exhibits an additional fast deactivation component that is assigned to an iminol tautomer. Reduced flavin cofactors also exhibit a long-lived component that is attributed to the semiquinone and the hydrated electron that are produced in photoinduced electron transfer to the solvent. The presence of adenine in FADH2 and FADH- further changes the excited-state dynamics due to intramolecular electron transfer from the isoalloxazine to the adenine moiety of cofactors. This electron transfer is more pronounced in FADH2 due to pi-stacking interactions between two moieties. We further studied cyclobutane thymine dimer (TT-dimer) repair via FADH- and FMNH- and found that the repair is much more efficient in the case of FADH-. These results suggest that the adenine moiety plays a significant role in the TT-dimer repair dynamics. Two possible explanations for the adenine mediation are presented: (i) a two-step electron transfer process, with the initial electron transfer occurring from flavin to adenine moiety of FADH-, followed by a second electron transfer from adenine to TT-dimer; (ii) the preconcentration of TT-dimer molecules around the flavin cofactor due to the hydrophobic nature of the adenine moiety.     

Cofactor modified electrodes

Studies are underway to see if flavin adenine dinucleotide or nicotinamide adenine dinucleotide cofactors can be attached to electrode surfaces to give rapid rates of electron transfer and in some cases reconstitution of enzyme activity with appropriate apoenzymes. Such electrodes may find widespread application in analytical chemistry.     

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.     

苹果酸酶结合域定点突变对烟酰胺腺嘌呤二核苷酸类似物催化性能的影响

通过对苹果酸酶(ME)辅酶结合域L310、Q401、L404饱和位点突变库与辅酶烟酰胺腺嘌呤二核苷酸(NAD⁺)类似物库的高通量筛选,研究了苹果酸酶结合域位点对NAD⁺及其类似物(B1~B7)催化活性的影响。 结果表明,突变后酶ME-Q401H/L404T对类似物B4的k_cat/K_m是野生型酶的50倍;突变后酶ME-L310M/Q401N对类似物B4的k_cat/K_m是野生型酶的16倍,对类似物B3的k_cat/K_m是野生型酶的5倍,因此通过对结合域定点突变,NAD⁺类似物的催化活性得到提高。     

Production of Hydroxy Acids: Selective Double Oxidation of Diols by Flavoprotein Alcohol Oxidase

Flavoprotein oxidases can catalyze oxidations of alcohols and amines by merely using molecular oxygen as the oxidant, making this class of enzymes appealing for biocatalysis. The FAD‐containing (FAD=flavin adenine dinucleotide) alcohol oxidase from P. chrysosporium facilitated double and triple oxidations for a range of aliphatic diols. Interestingly, depending on the diol substrate, these reactions result in formation of either lactones or hydroxy acids. For example, diethylene glycol could be selectively and fully converted into 2‐(2‐hydroxyethoxy)acetic acid. Such a facile cofactor‐independent biocatalytic route towards hydroxy acids opens up new avenues for the preparation of polyester building blocks.