Mesaconase/Fumarase FumD in Escherichia coli O157:H7 and Promiscuity of Escherichia coli Class I Fumarases FumA and FumB.
ABSTRACT: Mesaconase catalyzes the hydration of mesaconate (methylfumarate) to (S)-citramalate. The enzyme participates in the methylaspartate pathway of glutamate fermentation as well as in the metabolism of various C5-dicarboxylic acids such as mesaconate or L-threo-?-methylmalate. We have recently shown that Burkholderia xenovorans uses a promiscuous class I fumarase to catalyze this reaction in the course of mesaconate utilization. Here we show that classical Escherichia coli class I fumarases A and B (FumA and FumB) are capable of hydrating mesaconate with 4% (FumA) and 19% (FumB) of the catalytic efficiency kcat/Km, compared to the physiological substrate fumarate. Furthermore, the genomes of 14.8% of sequenced Enterobacteriaceae (26.5% of E. coli, 90.6% of E. coli O157:H7 strains) possess an additional class I fumarase homologue which we designated as fumarase D (FumD). All these organisms are (opportunistic) pathogens. fumD is clustered with the key genes for two enzymes of the methylaspartate pathway of glutamate fermentation, glutamate mutase and methylaspartate ammonia lyase, converting glutamate to mesaconate. Heterologously produced FumD was a promiscuous mesaconase/fumarase with a 2- to 3-fold preference for mesaconate over fumarate. Therefore, these bacteria have the genetic potential to convert glutamate to (S)-citramalate, but the further fate of citramalate is still unclear. Our bioinformatic analysis identified several other putative mesaconase genes and revealed that mesaconases probably evolved several times from various class I fumarases independently. Most, if not all iron-dependent fumarases, are capable to catalyze mesaconate hydration.
Project description: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 <i>Bacillus subtilis</i> class-II fumarase, in addition to its role in the tricarboxylic acid cycle, participates in the DDR. <i>Escherichia coli</i> harbors three fumarase genes: class-I <i>fumA</i> and <i>fumB</i> and class-II <i>fumC</i> Notably, class-I fumarases show no sequence similarity to class-II fumarases and are of different evolutionary origin. Strikingly, here we show that <i>E. coli</i> fumarase functions are distributed between class-I fumarases, which participate in the DDR, and the class-II fumarase, which participates in respiration. In <i>E. coli</i>, we discover that the signaling molecule, alpha-ketoglutarate (α-KG), has a function, complementing DNA damage sensitivity of <i>fum</i>-null mutants. Excitingly, we identify the <i>E. coli</i> 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 <i>fum</i>-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.
Project description:Pseudomonas aeruginosa, Yersinia pestis, and many other bacteria are able to utilize the C5-dicarboxylic acid itaconate (methylenesuccinate). Itaconate degradation starts with its activation to itaconyl coenzyme A (itaconyl-CoA), which is further hydrated to (S)-citramalyl-CoA, and citramalyl-CoA is finally cleaved into acetyl-CoA and pyruvate. The xenobiotic-degrading betaproteobacterium Burkholderia xenovorans possesses a P. aeruginosa-like itaconate degradation gene cluster and is able to grow on itaconate and its isomer mesaconate (methylfumarate). Although itaconate degradation proceeds in B. xenovorans in the same way as in P. aeruginosa, the pathway of mesaconate utilization is not known. Here, we show that mesaconate is metabolized through its hydration to (S)-citramalate. The latter compound is then metabolized to acetyl-CoA and pyruvate with the participation of two enzymes of the itaconate degradation pathway, a promiscuous itaconate-CoA transferase able to activate (S)-citramalate in addition to itaconate and (S)-citramalyl-CoA lyase. The first reaction of the pathway, the mesaconate hydratase (mesaconase) reaction, is catalyzed by a class I fumarase. As this enzyme (Bxe_A3136) has similar efficiencies (kcat/Km) for both fumarate and mesaconate hydration, we conclude that B. xenovorans class I fumarase is in fact a promiscuous fumarase/mesaconase. This promiscuity is physiologically relevant, as it allows the growth of this bacterium on mesaconate as a sole carbon and energy source.
Project description:<h4>Background</h4>The highly homologous [4Fe-4S] containing fumarases FumA and FumB, sharing 90% amino acid sequence identity, from Escherichia coli are differentially regulated, which suggests a difference in their physiological function. The ratio of FumB over FumA expression levels increases by one to two orders of magnitude upon change from aerobic to anaerobic growth conditions.<h4>Methodology/principal findings</h4>To understand this difference in terms of structure-function relations, catalytic and thermodynamic properties were determined for the two enzymes obtained from homologous overexpression systems. FumA and FumB are essentially identical in their Michaelis-Menten kinetics of the reversible fumarate to L-malate conversion; however, FumB has a significantly greater catalytic efficiency for the conversion of D-tartrate to oxaloacetate consistent with the requirement of the fumB gene for growth on D-tartrate. Reduction potentials of the [4Fe-4S](2+) Lewis acid active centre were determined in mediated bulk titrations in the presence of added substrate and were found to be approximately -290 mV for both FumA and FumB.<h4>Conclusions/significance</h4>This study contradicts previously published claims that FumA and FumB exhibit different catalytic preferences for the natural substrates L-malate and fumarate. FumA and FumB differ significantly only in the catalytic efficiency for the conversion of D-tartrate, a supposedly non-natural substrate. The reduction potential of the substrate-bound [4Fe-4S] active centre is, contrary to previously reported values, close to the cellular redox potential.
Project description:Growth on acetate or other acetyl-CoA-generating substrates as a sole source of carbon requires an anaplerotic pathway for the conversion of acetyl-CoA into cellular building blocks. Haloarchaea (class <i>Halobacteria</i>) possess two different anaplerotic pathways, the classical glyoxylate cycle and the novel methylaspartate cycle. The methylaspartate cycle was discovered in <i>Haloarcula</i> spp. and operates in ?40% of sequenced haloarchaea. In this cycle, condensation of one molecule of acetyl-CoA with oxaloacetate gives rise to citrate, which is further converted to 2-oxoglutarate and then to glutamate. The following glutamate rearrangement and deamination lead to mesaconate (methylfumarate) that needs to be activated to mesaconyl-C1-CoA and hydrated to ?-methylmalyl-CoA. The cleavage of ?-methylmalyl-CoA results in the formation of propionyl-CoA and glyoxylate. The carboxylation of propionyl-CoA and the condensation of glyoxylate with another acetyl-CoA molecule give rise to two C<sub>4</sub>-dicarboxylic acids, thus regenerating the initial acetyl-CoA acceptor and forming malate, its final product. Here we studied two enzymes of the methylaspartate cycle from <i>Haloarcula hispanica</i>, succinyl-CoA:mesaconate CoA-transferase (mesaconate CoA-transferase, Hah_1336) and mesaconyl-CoA hydratase (Hah_1340). Their genes were heterologously expressed in <i>Haloferax volcanii</i>, and the corresponding enzymes were purified and characterized. Mesaconate CoA-transferase was specific for its physiological substrates, mesaconate and succinyl-CoA, and produced only mesaconyl-C1-CoA and no mesaconyl-C4-CoA. Mesaconyl-CoA hydratase had a 3.5-fold bias for the physiological substrate, mesaconyl-C1-CoA, compared to mesaconyl-C4-CoA, and virtually no activity with other tested enoyl-CoA/3-hydroxyacyl-CoA compounds. Our results further prove the functioning of the methylaspartate cycle in haloarchaea and suggest that mesaconate CoA-transferase and mesaconyl-CoA hydratase can be regarded as characteristic enzymes of this cycle.
Project description:The nucleotide sequences of two segments of DNA (2250 and 2921 base-pairs) containing the functionally related fumarase (fumC) and aspartase (aspA) genes of Escherichia coli K12 were determined. The fumC structural gene comprises 1398 base-pairs (466 codons, excluding the initiation codon), and it encodes a polypeptide of Mr 50353 that resembles the fumarases of Bacillus subtilis 168 (citG-gene product), rat liver and pig heart. The fumC gene starts 140 base-pairs downstream of the structurally-unrelated fumA gene, but there is no evidence that both genes form part of the same operon. The aspA structural gene comprises 1431 base-pairs (477 codons excluding the initiation codon), and it encodes a polypeptide of Mr 52190, similar to that predicted from maxicell studies and for the enzyme from E. coli W. Remarkable homologies were found between the primary structures of the fumarase (fumC and citG) and aspartase (aspA) genes and their products, suggesting close structural and evolutionary relationships.
Project description:The tricarboxylic acid cycle produces NADH for oxidative phosphorylation and fumarase [EC 126.96.36.199] is a critical enzyme in this cycle, catalysing the reversible conversion of fumarate and L-malate. Fumarase is applied to industrial L-malate production as a biocatalyst. L-malate is used in a wide range of industries such as food and beverage, pharmacy chemistry. Although the biochemical properties of fumarases have been studied in many organisms, they have not been investigated in cyanobacteria. In this study, the optimum pH and temperature of Synechocystis 6803 fumarase C (SyFumC) were 7.5 and 30 °C, respectively. The Km of SyFumC for L-malate was higher than for fumarate. Furthermore, SyFumC activity was strongly inhibited by citrate and succinate, consistent with fumarases in other organisms. Substitution of alanine by glutamate at position 314 of SyFumC changed the kcat for fumarate and L-malate. In addition, the inhibitory effects of citrate and succinate on SyFumC activity were alleviated. Phylogenetic analysis revealed cyanobacterial fumarase clades divided in non-nitrogen-fixing cyanobacteria and nitrogen-fixing cyanobacteria. SyFumC was thus biochemically characterised, including identification of an amino acid residue important for substrate affinity and enzymatic activity.
Project description:Methylaspartate ammonia-lyase (MAL; EC 188.8.131.52) catalyzes the reversible addition of ammonia to mesaconate to yield l-threo-(2S,3S)-3-methylaspartate and l-erythro-(2S,3R)-3-methylaspartate as products. In the proposed minimal mechanism for MAL of Clostridium tetanomorphum, Lys-331 acts as the (S)-specific base catalyst and abstracts the 3S-proton from l-threo-3-methylaspartate, resulting in an enolate anion intermediate. This enolic intermediate is stabilized by coordination to the essential active site Mg(2+) ion and hydrogen bonding to the Gln-329 residue. Collapse of this intermediate results in the release of ammonia and the formation of mesaconate. His-194 likely acts as the (R)-specific base catalyst and abstracts the 3R-proton from the l-erythro isomer of 3-methylaspartate, yielding the enolic intermediate. In the present study, we have investigated the importance of the residues Gln-73, Phe-170, Gln-172, Tyr-356, Thr-360, Cys-361 and Leu-384 for the catalytic activity of C. tetanomorphum MAL. These residues, which are part of the enzyme surface lining the substrate binding pocket, were subjected to site-directed mutagenesis and the mutant enzymes were characterized for their structural integrity, ability to catalyze the amination of mesaconate, and regio- and diastereoselectivity. Based on the observed properties of the mutant enzymes, combined with previous structural studies and protein engineering work, we propose a detailed catalytic mechanism for the MAL-catalyzed reaction, in which the side chains of Gln-73, Gln-172, Tyr-356, Thr-360, and Leu-384 provide favorable interactions with the substrate, which are important for substrate binding and activation. This detailed knowledge of the catalytic mechanism of MAL can serve as a guide for future protein engineering experiments.
Project description:Methylaspartate ammonia lyase (MAL; EC 184.108.40.206) catalyzes the reversible addition of ammonia to mesaconate to give (2S,3S)-3-methylaspartate and (2S,3R)-3-methylaspartate as products. MAL is of considerable biocatalytic interest because of its potential use for the asymmetric synthesis of substituted aspartic acids, which are important building blocks for synthetic enzymes, peptides, chemicals, and pharmaceuticals. Here, we have cloned the gene encoding MAL from the thermophilic bacterium Carboxydothermus hydrogenoformans Z-2901. The enzyme (named Ch-MAL) was overproduced in Escherichia coli and purified to homogeneity by immobilized metal affinity chromatography. Ch-MAL is a dimer in solution, consisting of two identical subunits (?49 kDa each), and requires Mg(2+) and K(+) ions for maximum activity. The optimum pH and temperature for the deamination of (2S,3S)-3-methylaspartic acid are 9.0 and 70°C (k (cat)?=?78 s(-1) and K (m)?=?16 mM). Heat inactivation assays showed that Ch-MAL is stable at 50°C for >4 h, which is the highest thermal stability observed among known MALs. Ch-MAL accepts fumarate, mesaconate, ethylfumarate, and propylfumarate as substrates in the ammonia addition reaction. The enzyme also processes methylamine, ethylamine, hydrazine, hydroxylamine, and methoxylamine as nucleophiles that can replace ammonia in the addition to mesaconate, resulting in the corresponding N-substituted methylaspartic acids with excellent diastereomeric excess (>98% de). This newly identified thermostable MAL appears to be a potentially attractive biocatalyst for the stereoselective synthesis of aspartic acid derivatives on large (industrial) scale.
Project description: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.
Project description:The energy required for a bacterium to grow and colonize the host is generated by metabolic and respiratory functions of the cell. Proton motive force, produced by these processes, drives cellular mechanisms including redox balance, membrane potential, motility, acid resistance, and the import and export of substrates. Previously, disruption of succinate dehydrogenase (sdhB) and fumarate reductase (frdA) within the oxidative and reductive tricarboxylic acid (TCA) pathways in uropathogenic E. coli (UPEC) CFT073 indicated that the oxidative, but not the reductive TCA pathway, is required for fitness in the urinary tract. Those findings led to the hypothesis that fumA and fumC encoding fumarase enzymes of the oxidative TCA cycle would be required for UPEC colonization, while fumB of the reductive TCA pathway would be dispensable. However, only UPEC strains lacking fumC had a fitness defect during experimental urinary tract infection (UTI). To further characterize the role of respiration in UPEC during UTI, additional mutants disrupting both the oxidative and reductive TCA pathways were constructed. We found that knock-out of frdA in the sdhB mutant strain background ameliorated the fitness defect observed in the bladder and kidneys for the sdhB mutant strain and results in a fitness advantage in the bladder during experimental UTI. The fitness defect was restored in the sdhBfrdA double mutant by complementation with frdABCD. Taken together, we demonstrate that it is not the oxidative or reductive pathway that is important for UPEC fitness per se, but rather only the oxidative TCA enzyme FumC. This fumarase lacks an iron-sulfur cluster and is required for UPEC fitness during UTI, most likely acting as a counter measure against exogenous stressors, especially in the iron-limited bladder niche.