Enantioselectivity in the enzymatic dehydration of malate and tartrate: Mirror image specificities of structurally similar dehydratases.

Malate (2-hydroxysuccinic acid) and tartrate (2,3-dihydroxysuccinic acid) are chiral substrates; the former existing in two enantiomeric forms (R and S) while the latter exists as three stereoisomers (R,R; S,S; and R,S). Dehydration by stereospecific hydrogen abstraction and antielimination of the hydroxyl group yield the achiral products fumarate and oxaloacetate, respectively. Class-I fumarate hydratase (FH) and L-tartrate dehydratase (L-TTD) are two highly conserved enzymes belonging to the iron-sulfur cluster hydrolyase family of enzymes that catalyze reactions on specific stereoisomers of malate and tartrate. FH from Methanocaldococcus jannaschii accepts only (S)-malate and (S,S)-tartrate as substrates while the structurally similar L-TTD from Escherichia coli accepts only (R)-malate and (R,R)-tartrate as substrates. Phylogenetic analysis reveals a common evolutionary origin of L-TTDs and two-subunit archaeal FHs suggesting a divergence during evolution that may have led to the switch in substrate stereospecificity preference. Due to the high conservation of their sequences, a molecular basis for switch in stereospecificity is not evident from analysis of crystal structures of FH and predicted structure of L-TTD. The switch in enantiomer preference may be rationalized by invoking conformational plasticity of the amino acids interacting with the substrate, together with substrate reorientation and conformer selection about the C2C3 bond of the dicarboxylic acid substrates. Although classical models of enzyme-substrate binding are insufficient to explain such a phenomenon, the enantiomer superposition model suggests that a minor reorientation in the active site residues could lead to the switch in substrate stereospecificity.

Revisiting the Burden Borne by Fumarase: Enzymatic Hydration of an Olefin.

Fumarate hydratase (FH) is a remarkable catalyst that decreases the free energy of the catalyzed reaction by 30 kcal mol-1, much larger than most exceptional enzymes with extraordinary catalytic rates. Two classes of FH are observed in nature: class-I and class-II, which have different folds, yet catalyze the same reversible hydration/dehydration reaction of the dicarboxylic acids fumarate/malate, with equal efficiencies. Using class-I FH from the hyperthermophilic archaeon Methanocaldococcus jannaschii (Mj) as a model along with comparative analysis with the only other available class-I FH structure from Leishmania major (Lm), we provide insights into the molecular mechanism of catalysis in this class of enzymes. The structure of MjFH apo-protein has been determined, revealing that large intersubunit rearrangements occur across apo- and holo-protein forms, with a largely preorganized active site for substrate binding. Site-directed mutagenesis of active site residues, kinetic analysis, and computational studies, including density functional theory (DFT) and natural population analysis, together show that residues interacting with the carboxylate group of the substrate play a pivotal role in catalysis. Our study establishes that an electrostatic network at the active site of class-I FH polarizes the substrate fumarate through interactions with its carboxylate groups, thereby permitting an easier addition of a water molecule across the olefinic bond. We propose a mechanism of catalysis in FH that occurs through transition-state stabilization involving the distortion of the electronic structure of the substrate olefinic bond mediated by the charge polarization of the bound substrate at the enzyme active site.

Functional Characterization of FH Mutation c.557G>A Underlies Uterine Leiomyomas.

The FH gene encodes the fumarate hydratase of the Krebs cycle and functions as a homotetramer to catalyze the hydration of fumarate to malate. Mutations in FH result in uterine leiomyomas, a rare autosomal dominant inherited metabolic disease. However, how FH mutations result in this disease is poorly understood. Here, the FH mutation c.557G>A (p.S186N) was identified in a family with uterine leiomyomas phenotype. A series of studies were performed to confirm the pathogenicity of this mutation. Results showed that the FH mutant exhibited significantly lower fumarase enzyme activity and increased the fumarates level compared with the wildtype, which might be due to the impaired homotetramer formation in the native gel electrophoresis. Interestingly, the immunofluorescence study revealed that the overexpressed FH mutant exhibited puncta structures compared with the evenly expressed FH wildtype in cytoplasm suggesting that the altered amino acid might result in dysfunctional proteins which were accumulated to reduce its cytotoxicity. Importantly, the cells overexpressing the FH mutant exhibited higher proliferation and extracellular acidification rate value (ECAR) which might be caused by the upregulated HIF-1α indicating the tumor phenotype. Notably, phospho-mTOR was significantly increased and autophagy was inhibited in the FH mutant overexpression cells compared with the wildtype. Our work provides new insight into the FH mutation c.557G>A (p.S186N) underlies uterine leiomyomas and important information for accurate genetic counseling and clinical diagnosis of the disease.

Identification of zebrafish fumarate hydratase active site by molecular docking and simulation studies.

Fumarate hydratase (FH), one of the members of TCA cycle, acts as a catalyte for the synthesis of malate from fumarate. FH has been proposed to play as a tumour suppressor leading to the pathogenicity of leiomyomas, renal cell carcinoma and paraganglioma. Mutations in the active site of FH lead to alteration in the protein structure. Similarly, binding of several chemical inhibitors to the active site also leads to the disruption of protein structural integrity thereby leading to protein dysfunction. Therefore, in order to address this mechanism leading to cancer, the binding efficiency of potential human FH inhibitor citrate to zebrafish fh has been extensively analysed in this study by molecular docking and simulation experiments followed by quantification of fumarate hydratase enzyme activity to validate and confirm the findings. Molecular docking revealed stronger interaction of zebrafish fh protein with inhibitor citrate when compared to natural substrate fumarate. Study on the dynamics of docked structures further confirmed that citrate was found to possess more binding affinity than fumarate. In vitro biochemical analysis also revealed concentration dependent potential inhibitory effect of citrate on zebrafish fh, thus confirming the findings of the in-silico experiments.Communicated by Ramaswamy H. Sarma.

A combination of Class-I fumarases and metabolites (α-ketoglutarate and fumarate) signal the DNA damage response in Escherichia coli.

Class-II fumarases (fumarate hydratase, FH) are dual-targeted enzymes occurring in the mitochondria and cytosol of all eukaryotes. They are essential components in the DNA damage response (DDR) and, more specifically, protect cells from DNA double-strand breaks. Similarly, the gram-positive bacterium Bacillus subtilis class-II fumarase, in addition to its role in the tricarboxylic acid cycle, participates in the DDR. Escherichia coli harbors three fumarase genes: class-I fumA and fumB and class-II fumC Notably, class-I fumarases show no sequence similarity to class-II fumarases and are of different evolutionary origin. Strikingly, here we show that E. coli fumarase functions are distributed between class-I fumarases, which participate in the DDR, and the class-II fumarase, which participates in respiration. In E. coli, we discover that the signaling molecule, alpha-ketoglutarate (α-KG), has a function, complementing DNA damage sensitivity of fum-null mutants. Excitingly, we identify the E. coli α-KG-dependent DNA repair enzyme AlkB as the target of this interplay of metabolite signaling. In addition to α-KG, fumarate (fumaric acid) is shown to affect DNA damage repair on two different levels, first by directly inhibiting the DNA damage repair enzyme AlkB demethylase activity, both in vitro and in vivo (countering α-KG). The second is a more global effect on transcription, because fum-null mutants exhibit a decrease in transcription of key DNA damage repair genes. Together, these results show evolutionary adaptable metabolic signaling of the DDR, in which fumarases and different metabolites are recruited regardless of the evolutionary enzyme class performing the function.

Determination of glutathione-binding to proteins by fluorescence spectroscopy.

Glutathione (GSH) is the most abundant non-protein thiol and its cellular concentration has been reported as 17 mM in Escherichia coli. This study introduces a label-free method to determine the binding affinity of GSH to proteins, utilizing the intrinsic fluorescence of proteins; the dissociation constants of GSH for d-arabinose 5-phosphate isomerase KdsD, fumarase C, malate dehydrogenase, and RNA polymerase subunit α have been determined as 96 ± 8, 246 ± 42, 292 ± 78, and 296 ± 97 µM, respectively. The dissociation constants, less than 2% of the cellular concentration of GSH, suggests that protein-GSH interactions are strong enough to make all of the GSH-binding sites occupied fully. The method described here may be applicable to other proteins.

Post-translational Modifications of Fumarase Regulate its Enzyme Activity and Function in Respiration and the DNA Damage Response.

The Krebs cycle enzyme fumarase is a dual-targeted protein that is located in the mitochondria and cytoplasm of eukaryotic cells. Besides being involved in the TCA cycle and primary metabolism, fumarase is a tumour suppressor that aids DNA repair in human cells. Using mass spectrometry, we identified modifications in peptides of cytosolic yeast fumarase, some of which were absent when the cells were exposed to DNA damage (using the homing endonuclease system or hydroxyurea). We show that DNA damage increased the enzymatic activity of fumarase, which we hypothesized to be affected by post-translational modifications. Succinylation and ubiquitination of fumarase at lysines 78 and 79, phosphorylation at threonine 122, serine 124 and threonine 126 as well as deamidation at arginine 239 were found to be functionally relevant. Upon homology analysis, these residues were also found to be evolutionally conserved. Serine 128, on the other hand, is not evolutionary conserved and the Fum1S128D phosphorylation mimic was able to aid DNA repair. Our molecular model is that the above modifications inhibit the enzymatic activity of cytosolic fumarase under conditions of no DNA damage induction and when there is less need for the enzyme. Upon genotoxic stress, some fumarase modifications are removed and some enzymes are degraded while unmodified proteins are synthesized. This report is the first to demonstrate how post-translational modifications influence the catalytic and DNA repair functions of fumarase in the cell.

Closed fumarase C active-site structures reveal SS Loop residue contribution in catalysis.

Fumarase C (FumC) catalyzes the reversible conversion of fumarate to S-malate. Previous structural investigations within the superfamily have reported a dynamic structural segment, termed the SS Loop. To date, active-site asymmetry has raised the question of how SS Loop placement affects participation of key residues during the reaction. Herein, we report structural and kinetic analyses from Escherichia coli FumC variants to understand the contribution of SS Loop residues S318, K324, and N326. High-resolution X-ray crystallographic results reveal three distinct FumC active-site conformations; disordered-open, ordered-open, and the newly discovered ordered-closed. Surprisingly, each SS Loop variant has unaffected Michaelis constants coupled to reductions in turnover number. Based upon our structural and functional analyses, we propose structural and catalytic roles for each of the aforementioned residues.

Revisiting prochirality.

We propose a new model for prochirality that satisfies all known examples: the prochiral plane. This plane contains the prochiral carbon and defines two separate faces for chemical modification. We extend this to enzyme catalysis, replacing the "three point attachment" hypothesis and its variants. Once a prochiral substrate is fixed on an enzyme surface, the asymmetry of the enzyme provides reactants exclusively on one side of the prochiral plane, producing an enantiomerically pure chiral product. The aconitase reaction is detailed as an example, using molecular modeling and its known enzymatic mechanism. We show that the prochiral substrate for this enzyme is not citrate, but rather cis-aconitate. The number of interaction points of cis-aconitate is not relevant to prochirality, but rather to substrate specificity. A second detailed example is the enzyme fumarase; here the substrate fumarate has only two binding sites, but is nonetheless fixed onto the enzyme and has a defined prochiral plane. We also provide a literature survey of more prochiral substrates, all of which have sp2 hybridized carbon and contain a prochiral plane. An example of a prochiral unnatural substrate for sphingosine kinase 2, fingolimod, has an sp3 hybridized prochiral carbon and also contains a prochiral plane. Finally, we provide an intuitive example of a prochiral physical object, a coffee cup, interacting with one hand and lip.

Structural and Biochemical Investigations of the [4Fe-4S] Cluster-Containing Fumarate Hydratase from Leishmania major.

Class I fumarate hydratases (FHs) are central metabolic enzymes that use a [4Fe-4S] cluster to catalyze the reversible conversion of fumarate to S-malate. The parasite Leishmania major, which is responsible for leishmaniasis, expresses two class I FH isoforms: mitochondrial LmFH-1 and cytosolic LmFH-2. In this study, we present kinetic characterizations of both LmFH isoforms, present 13 crystal structures of LmFH-2 variants, and employ site-directed mutagenesis to investigate the enzyme's mechanism. Our kinetic data confirm that both LmFH-1 and LmFH-2 are susceptible to oxygen-dependent inhibition, with data from crystallography and electron paramagnetic resonance spectroscopy showing that oxygen exposure converts an active [4Fe-4S] cluster to an inactive [3Fe-4S] cluster. Our anaerobically conducted kinetic studies reveal a preference for fumarate over S-malate. Our data further reveal that single alanine substitutions of T467, R421, R471, D135, and H334 decrease kcat values 9-16000-fold without substantially affecting Km values, suggesting that these residues function in catalytic roles. Crystal structures of LmFH-2 variants are consistent with this idea, showing similar bidentate binding to the unique iron of the [4Fe-4S] cluster for substrate S-malate as observed in wild type FH. We further present LmFH-2 structures with substrate fumarate and weak inhibitors succinate and malonate bound in the active site and the first structure of an LmFH that is substrate-free and inhibitor-free, the latter showing increased mobility in the C-terminal domain. Collectively, these data provide insight into the molecular basis for the reaction catalyzed by LmFHs, enzymes that are potential drug targets against leishmaniasis.

A conserved motif liganding the [4Fe-4S] cluster in [4Fe-4S] fumarases prevents irreversible inactivation of the enzyme during hydrogen peroxide stress.

Organisms have evolved two different classes of the ubiquitous enzyme fumarase: the [4Fe-4S] cluster-containing class I enzymes are oxidant-sensitive, whereas the class II enzymes are iron-free and therefore oxidant-resistant. When hydrogen peroxide (H2O2) attacks the most-studied [4Fe-4S] fumarases, only the cluster is damaged, and thus the cell can rapidly repair the enzyme. However, this study shows that when elevated levels of H2O2 oxidized the class I fumarase of the obligate anaerobe Bacteroides thetaiotaomicron (Bt-Fum), a hydroxyl-like radical species was produced that caused irreversible covalent damage to the polypeptide. Unlike the fumarase of oxygen-tolerant bacteria, Bt-Fum lacks a key cysteine residue in the typical "CXnCX2C″ motif that ligands [4Fe-4S] clusters. Consequently H2O2 can access and oxidize an iron atom other than the catalytic one in its cluster. Phylogenetic analysis showed that certain clades of bacteria may have evolved the full "CXnCX2C″ motif to shield the [4Fe-4S] cluster of fumarase. This effect was reproduced by the construction of a chimeric enzyme. These data demonstrate the irreversible oxidation of Fe-S cluster enzymes and may recapitulate evolutionary steps that occurred when microorganisms originally confronted oxidizing environments. It is also suggested that, if H2O2 is generated within the colon as a consequence of inflammation or the action of lactic acid bacteria, the inactivation of fumarase could potentially impair the central fermentation pathway of Bacteroides species and contribute to gut dysbiosis.

Structural, biochemical and biophysical characterization of recombinant human fumarate hydratase.

Fumarate hydratases (FHs, fumarases) catalyze the reversible conversion of fumarate into l-malate. FHs are distributed over all organisms and play important roles in energy production, DNA repair and as tumor suppressors. They are very important targets both in the study of human metabolic disorders and as potential therapeutic targets in neglected tropical diseases and tuberculosis. In this study, human FH (HsFH) was characterized by using enzyme kinetics, differential scanning fluorimetry and X-ray crystallography. For the first time, the contribution of both substrates was analyzed simultaneously in a single kinetics assay allowing to quantify the contribution of the reversible reaction for kinetics. The protein was crystallized in the spacegroup C2221 , with unit-cell parameters a = 125.43, b = 148.01, c = 129.76. The structure was solved by molecular replacement and refined at 1.8 Å resolution. In our study, a HEPES molecule was found to interact with HsFH at the C-terminal domain (Domain 3), previously described as involved in allosteric regulation, through a set of interactions that includes Lys 467. HsFH catalytic efficiency is higher when in the presence of HEPES. Mutations at residue 467 have already been implicated in genetic disorders caused by FH deficiency, suggesting that the HEPES-binding site may be important for enzyme kinetics. This study contributes to the understanding of the HsFH structure and how it correlates with mutation, enzymatic deficiency and pathology.

Crystal Structures of Fumarate Hydratases from Leishmania major in a Complex with Inhibitor 2-Thiomalate.

Leishmaniases affect the poorest people on earth and have no effective drug therapy. Here, we present the crystal structure of the mitochondrial isoform of class I fumarate hydratase (FH) from Leishmania major and compare it to the previously determined cytosolic Leishmania major isoform. We further describe the mechanism of action of the first class-specific FH inhibitor, 2-thiomalate, through X-ray crystallography and inhibition assays. Our crystal structures of both FH isoforms with inhibitor bound at 2.05 Å resolution and 1.60 Å resolution show high structural similarity. These structures further reveal that the selectivity of 2-thiomalate for class I FHs is due to direct coordination of the inhibitor to the unique Fe of the catalytic [4Fe-4S] cluster that is found in class I parasitic FHs but is absent from class II human FH. These studies provide the structural scaffold in order to exploit class I FHs as potential drug targets against leishmaniases as well as Chagas diseases, sleeping sickness, and malaria.

Application of n-dodecane as an oxygen vector to enhance the activity of fumarase in recombinant Escherichia coli: role of intracellular microenvironment

Abstract The effect of the intracellular microenvironment in the presence of an oxygen vector during expression of a fusion protein in Escherichia coli was studied. Three organic solutions at different concentration were chosen as oxygen vectors for fumarase expression. The addition of n-dodecane did not induce a significant change in the expression of fumarase, while the activity of fumarase increased significantly to 124% at 2.5% n-dodecane added after 9 h induction. The concentration of ATP increased sharply during the first 6 h of induction, to a value 7600% higher than that in the absence of an oxygen-vector. NAD/NADH and NADP/NADPH ratios were positively correlated with fumarase activity. n-Dodecane can be used to increase the concentration of ATP and change the energy metabolic pathway, providing sufficient energy for fumarase folding.

Enhancing the thermostability of fumarase C from Corynebacterium glutamicum via molecular modification.

Fumarases have been successfully applied in industry for the production of l-malate. However, the industrialization of fumarases is limited by their low thermostability. In this study, the thermostability of fumarase C from Corynebacterium glutamicum was enhanced through directed evolution, simulated mutagenesis, site-directed mutagenesis and saturated mutagenesis. Mutant 2G (A411V) was initially constructed through directed evolution. Its half-life at 50 °C (t1/2, 50°C) increased from 1 min to 2.2 min, and the T5015 (temperature at which the activity of enzyme decreased by 50% in 15 min) increased from 44.8 °C to 47.2 °C. Besides, several different mutants were obtained by site-directed mutation. Among them, mutant 3G (A227V) showed significant improvement in thermostability with a 3.3-fold improvement of t1/2, 50°C and a 3.6 °C increase in T5015 compared to the wild-type enzyme. Then, 2/3G (A227V, A411V) was obtained by combining the mutant 2G with the mutant 3G, for which the t1/2, 50°C and T5015 increased to more than 768 min and 52.4 °C, respectively. Finally, site-saturated mutagenesis was employed on amino acid residues 175-Glu, 228-Gly, 297-Gly, 320-Lys and 464-Glu to maximize the thermostability of mutant 2/3G. The most thermostable mutant 175G with amino acid substitutions (A227V, A411V, E175K) was isolated. Its t1/2,50°C increased to more than 2700 min while that of wild-type enzyme was only 1 min and T5015 was 9.8 °C higher than the wild-type enzyme. The thermostable mutated enzymes generated without affecting the activity in this study would be an attractive candidate for industrial applications.

The complex allosteric and redox regulation of the fumarate hydratase and malate dehydratase reactions of Arabidopsis thaliana Fumarase 1 and 2 gives clues for understanding the massive accumulation of fumarate.

Arabidopsis thaliana possesses two fumarase genes (FUM), AtFUM1 (At2g47510) encoding for the mitochondrial Krebs cycle-associated enzyme and AtFUM2 (At5g50950) for the cytosolic isoform required for fumarate massive accumulation. Here, the comprehensive biochemical studies of AtFUM1 and AtFUM2 shows that they are active enzymes with similar kinetic parameters but differential regulation. For both enzymes, fumarate hydratase (FH) activity is favored over the malate dehydratase (MD) activity; however, MD is the most regulated activity with several allosteric activators. Oxalacetate, glutamine, and/or asparagine are modulators causing the MD reaction to become preferred over the FH reaction. Activity profiles as a function of pH suggest a suboptimal FUM activity in Arabidopsis cells; moreover, the direction of the FUM reaction is sensitive to pH changes. Under mild oxidation conditions, AtFUMs form high mass molecular aggregates, which present both FUM activities decreased to a different extent. The biochemical properties of oxidized AtFUMs (oxAtFUMs) were completely reversed by NADPH-supplied Arabidopsis leaf extracts, suggesting that the AtFUMs redox regulation can be accomplished in vivo. Mass spectrometry analyses indicate the presence of an active site-associated intermolecular disulfide bridge in oxAtFUMs. Finally, a phylogenetic approach points out that other plant species may also possess cytosolic FUM2 enzymes mainly encoded by paralogous genes, indicating that the evolutionary history of this trait has been drawn through a process of parallel evolution. Overall, according to our results, a multilevel regulatory pattern of FUM activities emerges, supporting the role of this enzyme as a carbon flow monitoring point through the organic acid metabolism in plants.

Application of n-dodecane as an oxygen vector to enhance the activity of fumarase in recombinant Escherichia coli: role of intracellular microenvironment.

The effect of the intracellular microenvironment in the presence of an oxygen vector during expression of a fusion protein in Escherichia coli was studied. Three organic solutions at different concentration were chosen as oxygen vectors for fumarase expression. The addition of n-dodecane did not induce a significant change in the expression of fumarase, while the activity of fumarase increased significantly to 124% at 2.5% n-dodecane added after 9h induction. The concentration of ATP increased sharply during the first 6h of induction, to a value 7600% higher than that in the absence of an oxygen-vector. NAD/NADH and NADP/NADPH ratios were positively correlated with fumarase activity. n-Dodecane can be used to increase the concentration of ATP and change the energy metabolic pathway, providing sufficient energy for fumarase folding.

Beyond the Michaelis-Menten equation: Accurate and efficient estimation of enzyme kinetic parameters.

Examining enzyme kinetics is critical for understanding cellular systems and for using enzymes in industry. The Michaelis-Menten equation has been widely used for over a century to estimate the enzyme kinetic parameters from reaction progress curves of substrates, which is known as the progress curve assay. However, this canonical approach works in limited conditions, such as when there is a large excess of substrate over enzyme. Even when this condition is satisfied, the identifiability of parameters is not always guaranteed, and often not verifiable in practice. To overcome such limitations of the canonical approach for the progress curve assay, here we propose a Bayesian approach based on an equation derived with the total quasi-steady-state approximation. In contrast to the canonical approach, estimates obtained with this proposed approach exhibit little bias for any combination of enzyme and substrate concentrations. Importantly, unlike the canonical approach, an optimal experiment to identify parameters with certainty can be easily designed without any prior information. Indeed, with this proposed design, the kinetic parameters of diverse enzymes with disparate catalytic efficiencies, such as chymotrypsin, fumarase, and urease, can be accurately and precisely estimated from a minimal amount of timecourse data. A publicly accessible computational package performing such accurate and efficient Bayesian inference for enzyme kinetics is provided.

Molecular docking and molecular dynamics simulations of fumarate hydratase and its mutant H235N complexed with pyromellitic acid and citrate.

Fumarase catalyzes the reversible, stereospecific hydration/dehydration of fumarate to L-malate during the Kreb's cycle. In the crystal structure of the tetrameric fumarase, it was found that some of the active site residues S145, T147, N188 G364 and H235 had water-mediated hydrogen bonding interactions with pyromellitic acid and citrate which help to the protonation state for the conversion of fumarate to malate. When His 235 is mutated with Asn (H235N), water-mediated interactions were lost due to the shifting of active site water molecule by 0.7 Å away. Molecular dynamics (MD) simulations were also carried out by NAMD and analyzed using Assisted Model Building with Energy Refinement (AMBER) program to better understand the conformational stability and other aspects during the binding of pyromellitic acid and citrate with native and mutant FH. The role of hydrogen bonds and hydrophobic interactions was also analyzed. The present study confirms that the H235N mutation has a major effect on the catalytic activity of fumarase which is evident from the biochemical studies.

Development of an Amperometric Biosensor Platform for the Combined Determination of L-Malic, Fumaric, and L-Aspartic Acid.

Three amperometric biosensors have been developed for the detection of L-malic acid, fumaric acid, and L -aspartic acid, all based on the combination of a malate-specific dehydrogenase (MDH, EC 1.1.1.37) and diaphorase (DIA, EC 1.8.1.4). The stepwise expansion of the malate platform with the enzymes fumarate hydratase (FH, EC 4.2.1.2) and aspartate ammonia-lyase (ASPA, EC 4.3.1.1) resulted in multi-enzyme reaction cascades and, thus, augmentation of the substrate spectrum of the sensors. Electrochemical measurements were carried out in presence of the cofactor ß-nicotinamide adenine dinucleotide (NAD+) and the redox mediator hexacyanoferrate (III) (HCFIII). The amperometric detection is mediated by oxidation of hexacyanoferrate (II) (HCFII) at an applied potential of + 0.3 V vs. Ag/AgCl. For each biosensor, optimum working conditions were defined by adjustment of cofactor concentrations, buffer pH, and immobilization procedure. Under these improved conditions, amperometric responses were linear up to 3.0 mM for L-malate and fumarate, respectively, with a corresponding sensitivity of 0.7 µA mM-1 (L-malate biosensor) and 0.4 µA mM-1 (fumarate biosensor). The L-aspartate detection system displayed a linear range of 1.0-10.0 mM with a sensitivity of 0.09 µA mM-1. The sensor characteristics suggest that the developed platform provides a promising method for the detection and differentiation of the three substrates.