The Involvement of Aspartate and Glutamate in the Decarboxylation of Malate by Isolated Bundle Sheath Chloroplasts from Zea mays

Aspartate or glutamate stimulated the rate of light-dependent malate decarboxylation by isolated Zea mays bundle sheath chloroplasts. Stimulation involved a decrease in the apparent K_m (malate) and an increased maximum velocity of decarboxylation. In the presence of glutamate other dicarboxylates (succinate, fumarate) competitively inhibited malate decarboxylation by intact chloroplasts with respect to malate with an apparent K_i of about 6 millimolar. For comparison the K_i for inhibition of nicotinamide adenine dinucleotide phosphate-malic enzyme from freshly lysed chloroplasts by these dicarboxylates was 15 millimolar. A range of compounds structurally related to aspartate stimulated malate decarboxylation by intact chloroplasts. K_a values for stimulation at 5 millimolar malate were 1.7, 5, and 10 millimolar for l-glutamate, l-aspartate, and β-methyl-dl-aspartate, respectively. Certain compounds, notably cysteic acid, which stimulated malate decarboxylation by intact chloroplasts inhibited malate decarboxylation by nicotinamide adenine dinucleotide phosphate-malic enzyme obtained from lysed chloroplasts and assayed under comparable conditions. It was concluded that aspartate, glutamate, and related compounds affect the transport of malate into the intact chloroplasts and that malate translocation does not take place on the general dicarboxylate translocator previously reported for higher plant chloroplasts.     

Anaerobic Accumulation of gamma-Aminobutyric Acid and Alanine in Radish Leaves (Raphanus sativus, L.)

In leaves, the anaerobic accumulation of alanine was accompanied by a loss of aspartate, and these changes preceded γ-aminobutyrate accumulation and glutamate loss. Changes in keto acid content did not appear to be the cause of amino acid changes. Accumulation of γ-aminobutyrate was due to acceleration of glutamate decarboxylation and arrest of γ-aminobutyrate transamination. Changes in enzyme content did not explain the changes in reaction rates in vivo. Most of the aspartate may be converted anaerobically to alanine via oxalacetate and pyruvate.     

Mechanism of asymmetric decarboxylation of α-aryl-α-methylmalonate catalyzed by arylmalonate decarboxylase originated from Alcaligenes bronchisepticus

Arylmalonate decarboxylase (EC. 4.1.1.76, originated from Alcaligenes bronchisepticus KU 1201) is an enzyme which catalyzes asymmetric decarboxylation of arylmalonate. We have once proposed the intermediary formation of a thiol ester between the substrate and the enzyme based on the inhibition studies with α-bromophenylacetate. We misinterpreted the binding mode of this acid as formation of thiol ester and estimated that the substrate also bound to the enzyme in the same manner. However, reinvestigation indicated that the mode of inhibition by this acid is irreversible, different from the previous conclusion. Accordingly the above mechanism became very unlikely. Instead, we would like to propose that Cys 188 is working as a proton donor on the basis of following evidence. The pH-rate of reaction profiles of the native and C188S mutant enzyme greatly differed in alkaline region. This is estimated to come from the difference in pK_a values of Cys and Ser, and suggested that Cys 188 is a proton donor. Homology alignment showed that this enzyme has some homology with glutamete racemase and some other isomerases. The presence of Cys 188 is conserved to all these enzymes as well as to AMDase. The role of this amino acid residue in glutamate racemase has been established to interchange a proton between the substrate. This fact also supports that Cys 188 of AMDase is working as a proton donor to form the asymmetric center of the product.     

Rapid Inactivation of Brain Glutamate Decarboxylase by Aspartate

In the absence of its cofactor, pyridoxal 5'-phosphate (pyridoxal-P), glutamate decarboxylase is rapidly inactivated by aspartate. Inactivation is a first-order process and the apparent rate constant is a simple saturation function of the concentration of aspartate. For the beta-form of the enzyme, the concentration of aspartate giving the half-maximal rate of inactivation is 6.1 +/- 1.3 mM and the maximal apparent rate constant is 1.02 +/- 0.09 min-1, which corresponds to a half-time of inactivation of 41 s. The rate of inactivation by aspartate is about 25 times faster than inactivation by glutamate or gamma-aminobutyric acid (GABA). Inactivation is accompanied by a rapid conversion of holoenzyme to apoenzyme and is opposed by pyridoxal-P, suggesting that inactivation results from an alternative transamination of aspartate catalyzed by the enzyme, as previously observed with glutamate and GABA. Consistent with this mechanism pyridoxamine 5'-phosphate, an expected transamination product, was formed when the enzyme was incubated with aspartate and pyridoxal-P. The rate of transamination relative to the rate of decarboxylation was much greater for aspartate than for glutamate. Apoenzyme formed by transamination of aspartate was reactivated with pyridoxal-P. In view of the high rate of inactivation, aspartate may affect the level of apoenzyme in brain.     

Aspartate stimulation of malate decarboxylation in Zea mays bundle sheath cells: possible role in regulation of C4 photosynthesis.

Aspartate stimulated by as much as three fold the rate of malate decarboxylation by Zea mays bundle sheath cells. Both the basal and aspartate stimulated rates of malate decarboxylation were light-dependent. Stimulation appeared to be due to aspartate as such, rather than depending on aspartate metabolism, and was due partly to a reduction in the malate concentration required for maximum decarboxylation and partly to an increased maximum velocity of decarboxylation. The extractable activities of NADP malic enzyme, glyceraldehyde phosphate dehydrogenase, and 3-phosphoglycerate kinase recoverable from cells were not increased by preincubating cells with aspartate, and aspartate did not affect the activity of these enzymes in cell-free extracts. It is suggested that aspartate may influence the transport of either malate into or pyruvate out of bundle sheath chloroplasts.     

Conversion of the catalytic specificity of alanine racemase to a -amino acid aminotransferase activity by a double active-site mutation

Alanine racemase depending on pyridoxal 5′-phosphate catalyzes the interconversion between - and -alanine. The enzyme from Bacillus stearothermophilus catalyzes the transamination as a side reaction with both substrates once per 3×10⁷ times of the racemization. In this work, we studied the effects of the mutation of Arg219, and that of Arg219 and Tyr265 on the catalysis of Bacillus alanine racemase. Arg219 interacting with pyridinium nitrogen of the cofactor is conserved in all alanine racemases. The corresponding residue of aminotransferases is an acidic residue, such as glutamate or aspartate. Mutation of Arg219 to a glutamyl residue resulted in a 5.4-fold increase in the forward half transamination activity with -alanine and a 10³-fold decrease in the racemase activity. The double mutation, Arg219→Glu and Tyr265→Ala, completely abolished the racemase activity and increased the forward half transaminase activity 6.6-fold. Arg219 is one of the structural determinants of the catalytic specificity of the alanine racemase.     

Substrate Shuttling between Active Sites of Uroporphyrinogen Decarboxylase Is Not Required to Generate Coproporphyrinogen

Uroporphyrinogen decarboxylase (URO-D; EC 4.1.1.37), the fifth enzyme of the heme biosynthetic pathway, is required for the production of heme, vitamin B12, siroheme, and chlorophyll precursors. URO-D catalyzes the sequential decarboxylation of four acetate side chains in the pyrrole groups of uroporphyrinogen to produce coproporphyrinogen. URO-D is a stable homodimer, with the active-site clefts of the two subunits adjacent to each other. It has been hypothesized that the two catalytic centers interact functionally, perhaps by shuttling of reaction intermediates between subunits. We tested this hypothesis by construction of a single-chain protein (single-chain URO-D) in which the two subunits were connected by a flexible linker. The crystal structure of this protein was shown to be superimposable with wild-type activity and to have comparable catalytic activity. Mutations that impaired one or the other of the two active sites of single-chain URO-D resulted in approximately half of wild-type activity. The distributions of reaction intermediates were the same for mutant and wild-type sequences and were unaltered in a competition experiment using I and III isomer substrates. These observations indicate that communication between active sites is not required for enzyme function and suggest that the dimeric structure of URO-D is required to achieve conformational stability and to create a large active-site cleft.     

An examination of aspartate decarboxylase and glutamate decarboxylase activity in mosquitoes

A major pathway of beta-alanine synthesis in insects is through the alpha-decarboxylation of aspartate, but the enzyme involved in the decarboxylation of aspartate has not been clearly defined in mosquitoes and characterized in any insect species. In this study, we expressed two putative mosquito glutamate decarboxylase-like enzymes of mosquitoes and critically analyzed their substrate specificity and biochemical properties. Our results provide clear biochemical evidence establishing that one of them is an aspartate decarboxylase and the other is a glutamate decarboxylase. The mosquito aspartate decarboxylase functions exclusively on the production of beta-alanine with no activity with glutamate. Likewise the mosquito glutamate decarboxylase is highly specific to glutamate with essentially no activity with aspartate. Although insect aspartate decarboxylase shares high sequence identity with glutamate decarboxylase, we are able to closely predict aspartate decarboxylase from glutamate decarboxylase based on the difference of their active site residues.     

Engineered hydrophobic pocket of (S)-selective arylmalonate decarboxylase variant by simultaneous saturation mutagenesis to improve catalytic performance

A bacterial arylmalonate decarboxylase (AMDase) catalyzes asymmetric decarboxylation of unnatural arylmalonates to produce optically pure (R)-arylcarboxylates without the addition of cofactors. Previously, we designed an AMDase variant G74C/C188S that displays totally inverted enantioselectivity. However, the variant showed a 20,000-fold reduction in activity compared with the wild-type AMDase. Further studies have demonstrated that iterative saturation mutagenesis targeting the active site residues in a hydrophobic pocket of G74C/C188S leads to considerable improvement in activity where all positive variants harbor only hydrophobic substitutions. In this study, simultaneous saturation mutagenesis with a restricted set of amino acids at each position was applied to further heighten the activity of the (S)-selective AMDase variant toward α-methyl-α-phenylmalonate. The best variant (V43I/G74C/A125P/V156L/M159L/C188G) showed 9,500-fold greater catalytic efficiency k_cat/K_m than that of G74C/C188S. Notably, a high level of decarboxylation of α-(4-isobutylphenyl)-α-methylmalonate by the sextuple variant produced optically pure (S)-ibuprofen, an analgesic compound which showed 2.5-fold greater activity than the (R)-selective wild-type AMDase.     

Pyruvate decarboxylation in astrocytes and in neurons in primary cultures in the presence and the absence of ammonia

Oxidative decarboxylation of [1-¹⁴C]pyruvate was studied in primary cultures of neurons and of astrocytes. The rate of this process, which is a measure of carbon flow into the tricarboxylic acid (TCA) cycle and which is inhibited by its end product, acetyl CoA, was determined under conditions which would either elevate or reduce the components of the malate-aspartate shuttle (MAS). Addition of aspartate (1 mM) was found to stimulate pyruvate decarboxylation in astrocytes whereas addition of glutamate (or glutamine) had no effect. Since aspartate is a precursor for extramitochondrial malate, and thus intramitochondrial oxaloacetate, whereas glutamate and glutamine are not, this suggests that an increase in oxaloacetate level stimulates TCA cycle activity. Conversely, a reduction of the glutamate content by 3 mM ammonia, which might reduce exchange between glutamate and aspartate across the mitochondrial membrane, suppressed pyruvate decarboxylation. This effect was abolished by addition of glutamate or glutamine or exposure to methionine sulfoximine (MSO). These findings suggest that impairment of MAS activity by removal of MAS constituents decreases TCA cycle activity whereas replenishment of these compounds restores the activity of the TCA cycle. No corresponding effects were observed in neurons.     

Structural characterization of the As/Sb reductase LmACR2 from Leishmania major

The arsenate/antimonate reductase LmACR2 has been recently identified in the genome of Leishmania major. Besides displaying phosphatase activity in vitro, this enzyme is able to reduce both As(V) and Sb(V) to their respective trivalent forms and is involved in the activation of Pentostan, a drug containing Sb(V) used in the treatment of leishmaniasis. LmACR2 displays sequence and functional similarity with the arsenate reductase ScACR2 from Saccharomyces cerevisiae, and both proteins are homologous to the catalytic domain of Cdc25 phosphatases, which, in turn, belong to the rhodanese/Cdc25 phosphatase superfamily. In this work, the three-dimensional structure of LmACR2 has been determined with crystallographic methods and refined at 2.15 Å resolution. The protein structure maintains the overall rhodanese fold, but substantial modifications are observed in secondary structure position and length. However, the conformation of the active-site loop and the position of the catalytic residue Cys75 are unchanged with respect to the Cdc25 phosphatases. From an evolutionary viewpoint, LmACR2 and the related arsenate reductases form, together with the known Cdc25 phosphatases, a well-defined subfamily of the rhodanese/Cdc25 phosphatase superfamily, characterized by a 7-amino-acid-long active-site loop that is able to selectively bind substrates containing phosphorous, arsenic, or antinomy. The evolutionary tree obtained for these proteins shows that, besides the active-site motif CE[F/Y]SXXR that characterizes Cdc25 phosphatase, the novel CALSQ[Q/V]R motif is also conserved in sequences from fungi and plants. Similar to Cdc25 phosphatase, these proteins are likely involved in cell cycle control. The active-site composition of LmACR2 (CAQSLVR) does not belong to either group, but gives to the enzyme a bifunctional activity of both phosphatase and As/Sb reductase. The subtle dependence of substrate specificity on the amino acid composition of the active-site loop displays the versatility of the ubiquitous rhodanese domain.     

Crystal structures of dialkylglycine decarboxylase inhibitor complexes.

The crystal structures of four inhibitor complexes of dialkylglycine decarboxylase are reported. The enzyme does not undergo a domain closure, as does aspartate aminotransferase, upon inhibitor binding. Two active-site conformations have been observed in previous structures that differ in alkali metal ion content, and two active-site conformations have been shown to coexist in solution when a single type of metal ion is present. There is no indication of coexisting conformers in the structures reported here or in the previously reported structures, and the observed conformation is that expected based on the presence of potassium in the enzyme. Thus, although two active-site conformations coexist in solution, a single conformation, corresponding to the more active enzyme, predominates in the crystal. The structure of 1-aminocyclopropane-1-carboxylate bound in the active site shows the aldimine double bond to the pyridoxal phosphate cofactor to be fully out of the plane of the coenzyme ring, whereas the Calpha-CO2(-) bond lies close to it. This provides an explanation for the observed lack of decarboxylation reactivity with this amino acid. The carboxylate groups of both 1-aminocyclopropane-1-carboxylate and 5'-phosphopyridoxyl-2-methylalanine interact with Ser215 and Arg406 as previously proposed. This demonstrates structurally that alternative binding modes, which constitute substrate inhibition, occur in the decarboxylation half-reaction. The structures of d and l-cycloserine bound to the active-site show that the l-isomer is deprotonated at C(alpha), presumably by Lys272, while the d-isomer is not. This difference explains the approximately 3000-fold greater potency of the l versus the d-isomer as a competitive inhibitor of dialkylglycine decarboxylase.     

An Archaeal Glutamate Decarboxylase Homolog Functions as an Aspartate Decarboxylase and Is Involved in β-Alanine and Coenzyme A Biosynthesis

β-Alanine is a precursor for coenzyme A (CoA) biosynthesis and is a substrate for the bacterial/eukaryotic pantothenate synthetase and archaeal phosphopantothenate synthetase. β-Alanine is synthesized through various enzymes/pathways in bacteria and eukaryotes, including the direct decarboxylation of Asp by aspartate 1-decarboxylase (ADC), the degradation of pyrimidine, or the oxidation of polyamines. However, in most archaea, homologs of these enzymes are not present; thus, the mechanisms of β-alanine biosynthesis remain unclear. Here, we performed a biochemical and genetic study on a glutamate decarboxylase (GAD) homolog encoded by TK1814 from the hyperthermophilic archaeon Thermococcus kodakarensis. GADs are distributed in all three domains of life, generally catalyzing the decarboxylation of Glu to γ-aminobutyrate (GABA). The recombinant TK1814 protein displayed not only GAD activity but also ADC activity using pyridoxal 5′-phosphate as a cofactor. Kinetic studies revealed that the TK1814 protein prefers Asp as its substrate rather than Glu, with nearly a 20-fold difference in catalytic efficiency. Gene disruption of TK1814 resulted in a strain that could not grow in standard medium. Addition of β-alanine, 4′-phosphopantothenate, or CoA complemented the growth defect, whereas GABA could not. Our results provide genetic evidence that TK1814 functions as an ADC in T. kodakarensis, providing the β-alanine necessary for CoA biosynthesis. The results also suggest that the GAD activity of TK1814 is not necessary for growth, at least under the conditions applied in this study. TK1814 homologs are distributed in a wide range of archaea and may be responsible for β-alanine biosynthesis in these organisms.     

Structural and Functional Analyses of β-Glucosidase 3B from Thermotoga neapolitana: A Thermostable Three-Domain Representative of Glycoside Hydrolase 3

Based on sequence and phylogenetic analyses, glycoside hydrolase (GH) family 3 can be divided into several clusters that differ in the length of their primary sequences. However, structural data on representatives of GH3 are still scarce, since only three of their structures are known and only one of them has been thoroughly characterized—that of an exohydrolase from barley. To allow a deeper structural understanding of the GH3 family, we have determined the crystal structure of the thermostable β-glucosidase from Thermotoga neapolitana, which has potentially important applications in environmentally friendly industrial biosynthesis at a resolution of 2.05 Å. Selected active-site mutants have been characterized kinetically, and the structure of the mutant D242A is presented at 2.1 Å resolution. Bgl3B from Th. neapolitana is the first example of a GH3 glucosidase with a three-domain structure. It is composed of an (α/β)₈ domain similar to a triose phosphate isomerase barrel, a five-stranded α/β sandwich domain (both of which are important for active-site organization), and a C-terminal fibronectin type III domain of unknown function. Remarkably, the direction of the second β-strand of the triose phosphate isomerase barrel domain is reversed, which has implications for the active-site shape. The active site, at the interface of domains 1 and 2, is much more open to solvent than the corresponding site in the structurally homologous enzyme from barley, and only the − 1 site is well defined. The structures, in combination with kinetic studies of active-site variants, allow the identification of essential catalytic residues (the nucleophile D242 and the acid/base E458), as well as other residues at the − 1 subsite, including D58 and W243, which, by mutagenesis, are shown to be important for substrate accommodation/interaction. The position of the fibronectin type III domain excludes a direct participation of this domain in the recognition of small substrates, although it may be involved in the anchoring of the enzyme on large polymeric substrates and in thermostability.     

The Crystal Structure of the Pseudomonas dacunhae Aspartate-β-Decarboxylase Dodecamer Reveals an Unknown Oligomeric Assembly for a Pyridoxal-5′-Phosphate-Dependent Enzyme

The Pseudomonas dacunhael-aspartate-β-decarboxylase (ABDC, aspartate 4-decarboxylase, aspartate 4-carboxylyase, E.C. 4.1.1.12) is a pyridoxal-5′-phosphate (PLP)-dependent enzyme that catalyzes the β-decarboxylation of l-aspartate to produce l-alanine and CO₂. This catalytically versatile enzyme is known to form functional dodecamers at its optimal pH and is thought to work in conjunction with an l-Asp/l-Ala antiporter to establish a proton gradient across the membrane that can be used for ATP biosynthesis. We have solved the atomic structure of ABDC to 2.35 Å resolution using single-wavelength anomalous dispersion phasing. The structure reveals that ABDC oligomerizes as a homododecamer in an unknown mode among PLP-dependent enzymes and has highest structural homology with members of the PLP-dependent aspartate aminotransferase subfamily. The structure shows that the ABDC active site is very similar to that of aspartate aminotransferase. However, an additional arginine side chain (Arg37) was observed flanking the re-side of the PLP ring in the ABDC active site. The mutagenesis results show that although Arg37 is not required for activity, it appears to be involved in the ABDC catalytic cycle.     

A novel combination of two classic catalytic schemes

The crystal structure of an alkaline Bacillus cellulase catalytic core, from glucoside hydrolase family 5, reveals a novel combination of the catalytic machinery of two classic textbook enzymes. The enzyme has the expected two glutamate residues in close proximity to one another in the active-site that are typical of retaining cellulases. However, the proton donor, glutamate 139 is also unexpectedly a member of a serine-histidine-glutamate catalytic triad, forming a novel combination of catalytic machineries. Structure and sequence analysis of glucoside hydrolase family 5 reveal that the triad is highly conserved, but with variations at the equivalent of the serine position. We speculate that the purpose of this novel catalytic triad is to control the protonation of the acid/base glutamate, facilitating the first step of the catalytic reaction, protonation of the substrate, by the proton donor glutamate. If correct, this will be a novel use for a catalytic triad.     

Crystal structures of isoorotate decarboxylases reveal a novel catalytic mechanism of 5-carboxyl-uracil decarboxylation and shed light on the search for DNA decarboxylase

DNA methylation and demethylation regulate many crucial biological processes in mammals and are linked to many diseases. Active DNA demethylation is believed to be catalyzed by TET proteins and a putative DNA decarboxylase that may share some similarities in sequence, structure and catalytic mechanism with isoorotate decarboxylase (IDCase) that catalyzes decarboxylation of 5caU to U in fungi. We report here the structures of wild-type and mutant IDCases from Cordyceps militaris and Metarhizium anisopliae in apo form or in complexes with 5caU, U, and an inhibitor 5-nitro-uracil. IDCases adopt a typical (β/α)₈ barrel fold of the amidohydrolase superfamily and function as dimers. A Zn²⁺ is bound at the active site and coordinated by four strictly conserved residues, one Asp and three His. The substrate is recognized by several strictly conserved residues. The functional roles of the key residues at the active site are validated by mutagenesis and biochemical studies. Based on the structural and biochemical data, we present for the first time a novel catalytic mechanism of decarboxylation for IDCases, which might also apply to other members of the amidohydrolase superfamily. In addition, our biochemical data show that IDCases can catalyze decarboxylation of 5caC to C albeit with weak activity, which is the first in vitro evidence for direct decarboxylation of 5caC to C by an enzyme. These findings are valuable in the identification of potential DNA decarboxylase in mammals.     

Pyruvate carboxylase from Mycobacterium smegmatis: stabilization,rapid purification,molecular and biochemical characterization and regulation of the cellular level

This is the first report on the purification and characterization of an anaplerotic enzyme from a Mycobacterium. The anaplerotic reactions play important roles in the biochemical differentiation of mycobacteria into non-replicating stages. We have purified and characterized a pyruvate carboxylase (PYC) from Mycobacterium smegmatis and cloned and sequenced its gene. We have developed a very rapid and efficient purification protocol that provided PYC with very high specific activities (up to 150 U/mg) that remained essentially unchanged over a month. The enzyme was found to be a homomultimer of 121 kDa subunits, mildly thermophilic, absolutely dependent on acyl-CoAs for activity and inhibited by ADP, by excess Mg²⁺, Co²⁺, and Mn²⁺, by aspartate, but not by glutamate and α-ketoglutarate. Supplementation of minimal growth medium with aspartate did not lower the cellular PYC level, rather doubled it; with glutamate the level remained unchanged. These observations would not fit the idea that the M. smegmatis enzyme fulfills a straightforward anaplerotic function; in a closely related organism, Corynebacterium glutamicum, PYC is the major anaplerotic enzyme. Growth on glucose provided 2-fold higher cellular PYC level than that observed with glycerol. The PYCs of M. smegmatis and Mycobacterium tuberculosis were highly homologous to each other. In M. smegmatis, M. tuberculosis and M. lepra, pyc was flanked by a putative methylase and a putative integral membrane protein genes in an identical operon-like arrangement. Thus, M. smegmatis could serve as a model for studying PYC-related physiological aspects of mycobacteria. Also, the ease of purification and the extraordinary stability could make the M. smegmatis enzyme a model for studying the structure–function relationships of PYCs in general. It should be noted that no crystal structure is available for this enzyme of paramount importance in all three domains of life, archaea, bacteria, and eukarya.     

Aspartate:2-oxoglutarate aminotransferase from Trichomonas vaginalis: comparison with pig heart cytoplasmic enzyme

The aspartate:2-oxoglutarate aminotransferase from the protozoon Trichomonas vaginalis exists as a mixture of sub-forms of identical Mr and amino acid composition, and of similar catalytic properties. The amino acid composition closely resembles that of aspartate aminotransferase from prokaryotic and vertebrate sources. Some molecular and catalytic properties of the T. vaginalis aspartate aminotransferase are compared with those of the cytoplasmic pig heart enzyme. A major difference is in the ability of the trichomonal enzyme to transaminate aromatic amino acids and 2-oxo acids. A range of inhibitors have been used to compare the active-site regions of the T. vaginalis and cytoplasmic pig heart aspartate aminotransferases.     

Crystal Structure and Putative Mechanism of 3-Methylitaconate-Δ-isomerase from Eubacterium barkeri

3-Methylitaconate-Δ-isomerase (Mii) participates in the nicotinate fermentation pathway of the anaerobic soil bacterium Eubacterium barkeri (order Clostridiales) by catalyzing the reversible conversion of (R)-3-methylitaconate (2-methylene-3-methylsuccinate) to 2,3-dimethylmaleate. The enzyme is also able to catalyze the isomerization of itaconate (methylenesuccinate) to citraconate (methylmaleate) with ca 10-fold higher K_m but > 1000-fold lower k_cat. The gene mii from E. barkeri was cloned and expressed in Escherichia coli. The protein produced with a C-terminal Strep-tag exhibited the same specific activity as the wild-type enzyme. The crystal structure of Mii from E. barkeri has been solved at a resolution of 2.70 Å. The asymmetric unit of the P2₁2₁2₁ unit cell with parameters a = 53.1 Å, b = 142.3 Å, and c = 228.4 Å contains four molecules of Mii. The enzyme belongs to a group of isomerases with a common structural feature, the so-called diaminopimelate epimerase fold. The monomer of 380 amino acid residues has two topologically similar domains exhibiting an α/β-fold. The active site is situated in a cleft between these domains. The four Mii molecules are arranged as a tetramer with 222 symmetry for the N-terminal domains. The C-terminal domains have different relative positions with respect to the N-terminal domains resulting in a closed conformation for molecule A and two distinct open conformations for molecules B and D. The C-terminal domain of molecule C is disordered. The Mii active site contains the putative catalytic residues Lys62 and Cys96, for which mechanistic roles are proposed based on a docking experiment of the Mii substrate complex. The active sites of Mii and the closely related PrpF, most likely a methylaconitate Δ-isomerase, have been compared. The overall architecture including the active-site Lys62, Cys96, His300, and Ser17 (Mii numbering) is similar. This positioning of (R)-3-methylitaconate allows Cys96 (as thiolate) to deprotonate C-3 and (as thiol) to donate a proton to the methylene carbon atom of the resulting allylic carbanion. Interestingly, the active site of isopentenyl diphosphate isomerase type I also contains a cysteine that cooperates with glutamate rather than lysine. It has been proposed that the initial step in this enzyme is a protonation generating a tertiary carbocation intermediate.