Identification of novel S-adenosyl-L-homocysteine hydrolase inhibitors through homology-model-based virtual screening, synthesis, and biological evaluation

The present study describes a successful application of computational approaches to identify novel Leishmania donovani (Ld) AdoHcyase inhibitors utilizing the differences for Ld AdoHcyase NAD(+) binding between human and Ld parasite. The development and validation of the three-dimensional (3D) structures of Ld AdoHcyase using the L. major AdoHcyase as template has been carried out. At the same time, cloning of the Ld AdoHcyase gene from clinical strains, its overexpression and purification have been performed. Further, the model was used in combined docking and molecular dynamics studies to validate the binding site of NAD in Ld. The hierarchical structure based virtual screening followed by the synthesis of five active hits and enzyme inhibition assay has resulted in the identification of novel Ld AdoHcyase inhibitors. The most potent inhibitor, compound 5, may serve as a "lead" for developing more potent Ld AdoHcy hydrolase inhibitors as potential antileishmanial agents.     

Protein conformational gating of enzymatic activity in xanthine oxidoreductase

In mammals, xanthine oxidoreductase can exist as xanthine dehydrogenase (XDH) and xanthine oxidase (XO). The two enzymes possess common redox active cofactors, which form an electron transfer (ET) pathway terminated by a flavin cofactor. In spite of identical protein primary structures, the redox potential difference between XDH and XO for the flavin semiquinone/hydroquinone pair (E(sq/hq)) is ~170 mV, a striking difference. The former greatly prefers NAD(+) as ultimate substrate for ET from the iron-sulfur cluster FeS-II via flavin while the latter only accepts dioxygen. In XDH (without NAD(+)), however, the redox potential of the electron donor FeS-II is 180 mV higher than that for the acceptor flavin, yielding an energetically uphill ET. On the basis of new 1.65, 2.3, 1.9, and 2.2 ? resolution crystal structures for XDH, XO, the NAD(+)- and NADH-complexed XDH, E(sq/hq) were calculated to better understand how the enzyme activates an ET from FeS-II to flavin. The majority of the E(sq/hq) difference between XDH and XO originates from a conformational change in the loop at positions 423-433 near the flavin binding site, causing the differences in stability of the semiquinone state. There was no large conformational change observed in response to NAD(+) binding at XDH. Instead, the positive charge of the NAD(+) ring, deprotonation of Asp429, and capping of the bulk surface of the flavin by the NAD(+) molecule all contribute to altering E(sq/hq) upon NAD(+) binding to XDH.     

Diphtheria toxin catalyzed hydrolysis of NAD(+): molecular dynamics study of enzyme-bound substrate, transition state, and inhibitor.

The mechanism of the diphtheria toxin-catalyzed hydrolysis of NAD(+) was investigated by quantum chemical calculations and molecular dynamics simulations. Several effects that could explain the 6000-fold rate acceleration (Delta Delta G(++) approximately 5 kcal/mol) by the enzyme were considered. First, the carboxamide arm of the enzyme-bound NAD(+) adopts a trans conformation while the most stable conformation is cis. The most stable conformation for the nicotinamide product has the amide carbonyl trans. The activation energy for the cleavage of the ribosidic bond is reduced by 2 kcal/mol due to the relaxation of this ground state conformational stress in the transition state. Second, molecular dynamics simulations to the nanosecond time range revealed that the carboxylate of Glu148 forms a hydrogen bond to the substrate's 2' hydroxyl group in E.S (approximately 17% of the time) and E.TS (approximately 57% of the time) complexes. This interaction is not seen in crystal structures. The ApUp inhibitor is held more tightly by the enzyme than the transition state and the substrate. Analysis of correlated motions reveals differences in the pattern of anticorrelated motions for protein backbone atoms when the transition state occupies the active site as compared to the E.NAD(+) complex.     

Nad Determination By Using A Glucose Dehydrogenase Based Glucose Probe

Abstract

This study describes the determination of NAD by using a glucose based enzyme carbon probe. Linear sweep voltammetric and constant potential studies were carried out in order to choose the best parameters for NAD analysis. The enzyme GDH has been used in solution first and then immobilized on the probe surface. Results indicate NAD can be detected in concentrations of 10^?6 Mol/1 with a good precision.     

Dynamic structures of horse liver alcohol dehydrogenase (HLADH): results of molecular dynamics simulations of HLADH-NAD(+)-PhCH(2)OH, HLADH-NAD(+)-PhCH(2)O(-), and HLADH-NADH-PhCHO.

Molecular dynamics simulations of the oxidation of benzyl alcohol by horse liver alcohol dehydrogenase (HLADH) have been carried out. The following three states have been studied: HLADH.PhCH(2)OH.NAD(+) (MD1), HLADH.PhCH(2)O(-).NAD(+) (MD2), and HLADH.PhCHO.NADH (MD3). MD1, MD2, and MD3 simulations were carried out on one of the subunits of the dimeric enzyme covered in a 32-A-radius sphere of TIP3P water centered on the active site. The proton produced on ionization of the alcohol when HLADH.PhCH(2)OH.NAD(+) --> HLADH.PhCH(2)O(-).NAD(+) is transferred from the active site to solvent water via a hydrogen bonding network consisting of serine48 hydroxyl, ribose 2'- and 3'-hydroxyl groups, and Hist51. Hydrogen bonding of the 3'OH of ribose to Ile269 carbonyl maintains this proton in position to be transferred to water. Molecular dynamic simulations have been employed to track water1287 from the TIP3 water pool to the active site, thus exhibiting the mode of entrance of water to the active site. With time the water1287 accumulates in two different positions in order to accept the proton from the ribose 3'-OH and from His51. There can be identified two structural substates for proton passage. In the first substate the imidazole Ne2 of His51 is adjacent to the nicotinamide ribose C2'-OH and hydrogen bonding distances for proton transfer through the hydrogen bonded relay series PhCH(2)OH...Ser48-OH...Ribose2'-OH...His51...OH(2) (path 1) average 2.0, 2.0, and 2.1 A and (for His51...OH(2)) minimal distances less or equal to 2.5 A. The structure for path 1 is present 20% of the time span. And in the second substate, there are two possible proton passages: path 1 as before and path 2. Path 2 involves the hydrogen-bonded relay series PhCH(2)OH...Ser48-OH...Ribose2'-OH...Ribose3'-OH...His51.OH(2) with the average bonding distances being 2.0, 2.0, 2.1, and 2.0 A and (for His51...OH(2)) minimal distances less or equal to 2.5 A (20% probability of the time span), respectively. During the molecular dynamics simulation the NAD(+) ribose conformations have stabilized at the C2'-endo-C3'-exo or the C2'-endo conformations. With the C2'-endo conformation the first and second substates are able to persist for different time spans, while with the C2'-endo-C3'-exo conformation the only possible pathway involves the first substate. For both first and second substates the fluctuation of the distances between the ribose-OH protons and N epsilon 2 of His51 imidazole ring is partially contributed by the "windshield wiper" motion of the His51 imidazole ring. Since the imidazole of His-51 contributes only about 10-fold to activity, as estimated from the decrease in activity upon substitution with a Gln, there must be an alternate route for the proton to pass to solvent without going through this histidine. A third pathway involves ribose C3'-OH and Ile-269. In MD2, near attack conformers (NACs) for hydride transfer from PhCH(2)O(-) to NAD(+) represent approximately 60% of E.S conformers. The molecular dynamic study of MD3 at mildly basic pH reveals that reactive ground state conformers (NACs) for hydride transfer from NADH to PhCHO amount to 12 mol % of conformers. In MD3, anisotropic bending of the dihydronicotinamide ring of NADH (average value of alpha(c) = 4.0 degrees and alpha(n) = 0.5 degrees, respectively) is observed.     

Ultrafast infrared spectroscopy reveals water-mediated coherent dynamics in an enzyme active site

Understanding the impact of fast dynamics upon the chemical processes occurring within the active sites of proteins and enzymes is a key challenge that continues to attract significant interest, though direct experimental insight in the solution phase remains sparse. Similar gaps in our knowledge exist in understanding the role played by water, either as a solvent or as a structural/dynamic component of the active site. In order to investigate further the potential biological roles of water, we have employed ultrafast multidimensional infrared spectroscopy experiments that directly probe the structural and vibrational dynamics of NO bound to the ferric haem of the catalase enzyme from Corynebacterium glutamicum in both H₂O and D₂O. Despite catalases having what is believed to be a solvent-inaccessible active site, an isotopic dependence of the spectral diffusion and vibrational lifetime parameters of the NO stretching vibration are observed, indicating that water molecules interact directly with the haem ligand. Furthermore, IR pump–probe data feature oscillations originating from the preparation of a coherent superposition of low-frequency vibrational modes in the active site of catalase that are coupled to the haem ligand stretching vibration. Comparisons with an exemplar of the closely-related peroxidase enzyme family shows that they too exhibit solvent-dependent active-site dynamics, supporting the presence of interactions between the haem ligand and water molecules in the active sites of both catalases and peroxidases that may be linked to proton transfer events leading to the formation of the ferryl intermediate Compound I. In addition, a strong, water-mediated, hydrogen bonding structure is suggested to occur in catalase that is not replicated in peroxidase; an observation that may shed light on the origins of the different functions of the two enzymes.     

Nitroxides as metabolic and EPR imaging probes in biological model systems

Nitroxides are unusually stable free radicals and sensitive “reporters” of the chemico-physical environment which surround them. The term “reporter group” was used to indicate this property, later the term spin label or spin probe was preferred. The facile synthesis and the multiplicity of compounds which have been coupled to the nitroxide moiety has allowed the development of a number of applications. Nitroxides have been used to study molecular mobility of proteins and lipids in membranes, enzyme active sites, DNA synthesis, and measure of electrical potential, pH, temperature as well as oxygen gradients across membranes; more recently, as contrast agents in magnetic resonance imaging and electron paramagnetic resonance imaging (EPRI). Aim of this work is to review and comment on the use of various nitroxides as metabolic probes, and in EPRI in studies of biomedical interest.     

Use of 6‐Methylisoxanthopterin,a Fluorescent Guanine Analog,to Probe Fob1‐Mediated Dynamics at the Stalling Fork Barrier DNA Sequences

Fluorescent nucleic acid base mimics serve as excellent site‐specific and real‐time reporters of the local and global dynamics. In this work, using the fluorescent guanine mimic 6‐methylisoxanthopterin (6‐MI), we unravel the differential dynamics of replication fork barrier/terminator sequences (RFB1 and RFB3) mediated by fork blocking protein (Fob1). By strategic and site‐specific incorporation of this probe, we show that 6‐MI is able to capture the changes in global dynamics exhibited by Fob1 and aids in distinguishing between varied architectural forms like double‐stranded DNA versus Holliday junctions (HJs). This is important as these barriers are hotspots for recombination. Fluorescence lifetime and anisotropy decay studies further revealed that Fob1 strongly dampens the dynamics in double‐stranded RFB1, and the sequence inherently possesses lesser flexibility in comparison to RFB3. We show that 6‐MI can probe the differential oligomeric status of Fob1 in response to various architectures, that is, double‐stranded versus HJs. This work highlights the unique advantages of 6‐MI as a probe when incorporated in nucleic acid frameworks.     

Full protein flexibility is essential for proper hot-spot mapping

A traditional technique for structure-based drug design (SBDD) is mapping of protein surfaces with probe molecules to identify "hot spots" where key functional groups can best complement the receptor. Common methods, such as minimization of probes or calculation of grids, use a fixed protein structure in the gas phase, ignoring both protein flexibility and proper competition between the probes and water. As a result, the potential surface is quite rugged, and many spurious local minima are identified. In this work, we compared rigid and fully flexible proteins in mixed-solvent molecular dynamics, which allows for flexibility and full solvent effects. We were surprised to find that the large number of local minima are still found when a protein's conformational sampling is restricted; the dynamic averaging of probes and competition with water do not smooth the potential surface as one might expect. Only when a protein is allowed to be fully flexible in the simulation are the proper minima located and the spurious ones eliminated. Our results indicate that inclusion of full protein flexibility is critical to accurate hot-spot mapping for SBDD.     

Factors controlling the mechanism of NAD(+) non-redox reactions

β-Nicotinamide adenine dinucleotide (NAD(+)) is an indispensable coenzyme or substrate for enzymes involved in catalyzing redox and non-redox reactions. ADP-ribosylating enzymes catalyze cleavage of the nicotinamide-glycosyl bond of NAD(+) and addition of a nucleophilic group from their substrate proteins to the N-ribose anomeric carbon of NAD(+). Although the role of the nicotinamide-ribose fragment in the mechanism of NAD(+) hydrolysis has been examined, the role of the doubly negatively charged, flexible, and chemically reactive NAD(+) diphosphate moiety in the reaction process has largely been neglected. Thus, the participation of the pyrophosphate group in stabilizing intra- and intermolecular interactions in the ground state and transition state has not been explored. Furthermore, the roles of other factors such as the type/nucleophilicity of the attacking nucleophile and the medium in influencing the reaction pathway have not been systematically evaluated. In this study, we endeavor to fill in these gaps and elucidate the role of these factors in controlling the NAD(+) nicotinamide-glycosyl bond cleavage. Using density functional theory combined with continuum dielectric methods, we modeled both S(N)1 and S(N)2 reaction pathways and assessed the role of the diphosphate group in stabilizing the (i) NAD(+) ground state, (ii) oxocarbocation intermediate, (iii) reaction product, and (iv) nucleophile. We also assessed the chemical nature of the attacking nucleophile and the role of the protein matrix in affecting the reaction mechanism. Our results reveal an intricate interplay among various factors in controlling the reaction pathway, which in turn suggests ways in which the enzyme can accelerate the reaction.     

The structure of NADH in the enzyme dTDP-d-glucose dehydratase (RmlB)

The structure of Streptococcus suis serotype type 2 dTDP-d-glucose 4,6-dehydratase (RmlB) has been determined to 1.5 A resolution with its nicotinamide coenzyme and substrate analogue dTDP-xylose bound in an abortive complex. During enzyme turnover, NAD(+) abstracts a hydride from the C4' atom of dTDP-glucose-forming NADH. After elimination of water, hydride is then transferred back to the C6' atom of dTDP-4-keto-5,6-glucosene-regenerating NAD(+). Single-crystal spectroscopic studies unambiguously show that the coenzyme has been trapped as NADH in the crystal. Electron density clearly demonstrates that in contrast to native structures of RmlB where a flat nicotinamide ring is observed, the dihydropyridine ring of the reduced cofactor in this complex is found as a boat. The si face, from which the pro-S hydride is transferred, has a concave surface. Ab initio electronic structure calculations demonstrate that the presence of an internal hydrogen bond, between the amide NH on the nicotinamide ring and one of the oxygen atoms on a phosphate group, stabilizes this distorted conformation. Additionally, calculations show that the hydride donor ability of NADH is influenced by the degree of bending in the ring and may be influenced by an active-site tyrosine residue (Tyr 161). These results demonstrate the ability of dehydratase enzymes to fine-tune the redox potential of NADH through conformational changes in the nicotinamide ring.     

Effect of the R119G mutation on human P5CR structure and its interactions with NAD: Insights derived from molecular dynamics simulation and free energy analysis

Pyrroline-5-carboxylate reductase (P5CR), an enzyme with conserved housekeeping roles, is involved in the etiology of cutis laxa. While previous work has shown that the R119G point mutation in the P5CR protein is involved, the structural mechanism behind the pathology remains to be elucidated. In order to probe the role of the R119G mutation in cutis laxa, we performed molecular dynamics (MD) simulations, essential dynamics (ED) analysis, and Molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) binding free energy calculations on wild type (WT) and mutant P5CR-NAD complex. These MD simulations and ED analyses suggest that the R119G mutation decreases the flexibility of P5CR, specifically in the substrate binding pocket, which could decrease the kinetics of the cofactor entrance and egress. Furthermore, the MM-PBSA calculations suggest the R119G mutant has a lower cofactor binding affinity for NAD than WT. Our study provides insight into the possible role of the R119G mutation during interactions between P5CR and NAD, thus bettering our understanding of how the mutation promotes cutis laxa.     

Design, synthesis and biological characterization of novel inhibitors of CD38

Human CD38 is a novel multi-functional protein that acts not only as an antigen for B-lymphocyte activation, but also as an enzyme catalyzing the synthesis of a Ca(2+) messenger molecule, cyclic ADP-ribose, from NAD(+). It is well established that this novel Ca(2+) signaling enzyme is responsible for regulating a wide range of physiological functions. Based on the crystal structure of the CD38/NAD(+) complex, we synthesized a series of simplified N-substituted nicotinamide derivatives (Compound 1-14). A number of these compounds exhibited moderate inhibition of the NAD(+) utilizing activity of CD38, with Compound 4 showing the highest potency. The crystal structure of CD38/Compound 4 complex and computer simulation of Compound 7 docking to CD38 show a significant role of the nicotinamide moiety and the distal aromatic group of the compounds for substrate recognition by the active site of CD38. Biologically, we showed that both Compounds 4 and 7 effectively relaxed the agonist-induced contraction of muscle preparations from rats and guinea pigs. This study is a rational design of inhibitors for CD38 that exhibit important physiological effects, and can serve as a model for future drug development.     

Is the mobility of the pore walls and water molecules in the selectivity filter of KcsA channel functionally important?

We performed in-depth analysis of the forces which act on the K(+) ions in the selectivity filter of the KcsA channel in order to estimate the relative importance of static and dynamic influence of the filter wall and water molecules on ion permeation and selectivity. The forces were computed using the trajectories of all-atom molecular dynamics simulations. It is shown that the dynamics of the selectivity filter contributes about 3% to the net force acting on the ions and can be neglected in the studies focused on the macroscopic properties of the channel, such as the current. Among the filter atoms, only the pore-forming carbonyl groups can be considered as dynamic in the studies of microscopic events of conduction, while the dynamic effects from all other atoms are negligible. We also show that the dynamics of the water molecules in the filter can not be neglected. The fluctuating forces from the water molecules can be as strong as net forces from the pore walls and can effectively drive the ions through the local energy barriers in the filter.     

Molecular clip and tweezer introduce new mechanisms of enzyme inhibition

Artificial molecular clips and tweezers, designed for cofactor and amino acid recognition, are able to inhibit the enzymatic activity of alcohol dehydrogenase (ADH). IC50 values and kinetic investigations point to two different new mechanisms of interference with the NAD(+)-dependent oxidoreductase: While the clip seems to pull the cofactor out of its cleft, the tweezer docks onto lysine residues around the active site. Both modes of action can be reverted to some extent, by appropriate additives. However, while cofactor depletion by clip 1 was in part restored by subsequent NAD(+) addition, the tweezer (2) inhibition requires the competitive action of lysine derivatives. Lineweaver-Burk plots indicate a competitive mechanism for the clip, with respect to both substrate and cofactor, while the tweezer clearly follows a noncompetitive mechanism. Conformational analysis by CD spectroscopy demonstrates significant ADH denaturation in both cases. However, only the latter case (tweezer-lysine) is reversible, in full agreement with the above-detailed enzyme switch experiments. The complexes of ADH with clips or tweezer can be visualized in a nondenaturing gel electrophoresis, where the complexes migrate toward the anode, in contrast to the pure enzyme which approaches the cathode. Supramolecular chemistry has thus been employed as a means to control protein function with the specificity of artificial hosts opening new avenues for this endeavor.     

Computer simulations of trypanosomal nucleoside hydrolase: determination of the protonation state of the bound transition-state analogue

Inosine-uridine nucleoside hydrolase (IU-NH) catalyzes the hydrolysis of nucleosides into base and ribose moieties via a ribooxocarbenium ion transition state, which has been characterized using kinetic isotope effects. Protozoan parasites lack de novo purine and pyrimidine biosynthesis and depend on the purine salvage from the host. Vern Schramm and co-workers characterized p-aminophenyliminoribitol (pAPIR) to be a potent inhibitor of IU-NH from Crithidia fasciculata with K(d) of 30 nM. The cyclic amine function of the iminoribitol ring can be either protonated (pAPIRH(+)) or unprotonated (pAPIR). pAPIRH(+) resembles the charge and geometry of the ribooxocarbenium ion transition state and can be looked upon as a transition-state analogue inhibitor; however, it is known that the pAPIR species is initially bound to the enzyme. We have characterized the pAPIRH(+) species as resident of the active site using ab initio calculations and molecular dynamics simulations. This is a novel use of molecular dynamics to investigate the protonation state of the bound ligand to the active site. Nanosecond molecular dynamics simulations reveal a short hydrogen-bonding network between pAPIRH(+)-O2'-Asp14-His241 triad, which is not seen in the crystal structure. Other features discussed are: hydrogen bonding between pAPIRH(+) and Asn168, unusual geometry of the iminoribitol ring, and hydrophobic interactions.     

Water dynamics and proton transfer in nafion fuel cell membranes

The dynamics of water and its effect on proton transport kinetics in Nafion membranes are compared at several hydration levels. Nafion is the most widely used polyelectrolyte membrane in fuel cells. Ultrafast infrared spectroscopy of the O-D stretch of dilute HOD in H2O provides a probe of the local environment and hydrogen bond network dynamics of water confined in the hydrophilic regions of Nafion. The kinetics of proton transfer in Nafion are tracked by following the excited-state proton transfer and recombination kinetics of a molecular probe, pyranine (HPTS). The hydrophilic domains of Nafion grow with increased hydration, and the interfacial regions reorganize, leading to a changing local environment for water near the interface. Swelling is not uniform throughout the membrane, and heterogeneity is observed in the fluorescence anisotropy decays of the methoxy derivative of pyranine. Measurements of the time-dependent anisotropy of water in Nafion provide a direct probe of the hydrogen bond network dynamics. These dynamics, as well as the rate of proton transport over nanoscopic distances, are observed to slow significantly as the hydration level of the membrane decreases. The results provide insights into the influence of changes in the dynamics of water on the proton-transfer processes.     

Reversible,electrochemical interconversion of NADH and NAD+ by the catalytic (Ilambda) subcomplex of mitochondrial NADH:ubiquinone oxidoreductase (complex I)

NADH:ubiquinone oxidoreductase (complex I) is the first enzyme of the mitochondrial electron transport chain and catalyzes the oxidation of beta-NADH by ubiquinone, coupled to transmembrane proton translocation. It contains a flavin mononucleotide (FMN) at the active site for NADH oxidation, up to eight iron-sulfur (FeS) clusters, and at least one ubiquinone binding site. Little is known about the mechanism of coupled electron-proton transfer in complex I. This communication demonstrates how the catalytic fragment of complex I, subcomplex Ilambda, can be adsorbed onto a pyrolytic graphite edge electrode to catalyze the interconversion of NADH and NAD+, with the electrode as the electron acceptor or donor. NADH oxidation and NAD+ reduction are completely reversible and occur without the application of an overpotential. The potential of zero current denotes the potential of the NAD+/NADH redox couple, and the dependence of ENAD+ on pH, and on the NADH:NAD+ ratio, is in accordance with the Nernst equation. The catalytic potential of the enzyme, Ecat, is close to one of the two reduction potentials of the active site FMN and to the potential of a nearby [2Fe - 2S] cluster; therefore, either one or both of these redox couples is suggested to be important in controlling NADH oxidation by complex I.     

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).