Oxidation of propionate to pyruvate in Escherichia coli. Involvement of methylcitrate dehydratase and aconitase.

The pathway of the oxidation of propionate to pyruvate in Escherichia coli involves five enzymes, only two of which, methylcitrate synthase and 2-methylisocitrate lyase, have been thoroughly characterized. Here we report that the isomerization of (2S,3S)-methylcitrate to (2R,3S)-2-methylisocitrate requires a novel enzyme, methylcitrate dehydratase (PrpD), and the well-known enzyme, aconitase (AcnB), of the tricarboxylic acid cycle. AcnB was purified as 2-methylaconitate hydratase from E. coli cells grown on propionate and identified by its N-terminus. The enzyme has an apparent Km of 210 micro m for (2R,3S)-2-methylisocitrate but shows no activity with (2S,3S)-methylcitrate. On the other hand, PrpD is specific for (2S,3S)-methylcitrate (Km = 440 micro m) and catalyses in addition only the hydration of cis-aconitate at a rate that is five times lower. The product of the dehydration of enzymatically synthesized (2S,3S)-methylcitrate was designated cis-2-methylaconitate because of its ability to form a cyclic anhydride at low pH. Hence, PrpD catalyses an unusual syn elimination, whereas the addition of water to cis-2-methylaconitate occurs in the usual anti manner. The different stereochemistries of the elimination and addition of water may be the reason for the requirement for the novel methylcitrate dehydratase (PrpD), the sequence of which seems not to be related to any other enzyme of known function. Northern-blot experiments showed expression of acnB under all conditions tested, whereas the RNA of enzymes of the prp operon (PrpE, a propionyl-CoA synthetase, and PrpD) was exclusively present during growth on propionate. 2D gel electrophoresis showed the production of all proteins encoded by the prp operon during growth on propionate as sole carbon and energy source, except PrpE, which seems to be replaced by acetyl-CoA synthetase. This is in good agreement with investigations on Salmonella enterica LT2, in which disruption of the prpE gene showed no visible phenotype.     

In vitro conversion of propionate to pyruvate by Salmonella enterica enzymes: 2-methylcitrate dehydratase (PrpD) and aconitase Enzymes catalyze the conversion of 2-methylcitrate to 2-methylisocitrate

Salmonella enterica serovar Typhimurium LT2 catabolizes propionate through the 2-methylcitric acid cycle, but the identity of the enzymes catalyzing the conversion of 2-methylcitrate into 2-methylisocitrate is unclear. This work shows that the prpD gene of the prpBCDE operon of this bacterium encodes a protein with 2-methylcitrate dehydratase enzyme activity. Homogeneous PrpD enzyme did not contain an iron-sulfur center, displayed no requirements for metal cations or reducing agents for activity, and did not catalyze the hydration of 2-methyl-cis-aconitate to 2-methylisocitrate. It was concluded that the gene encoding the 2-methyl-cis-aconitate hydratase enzyme is encoded outside the prpBCDE operon. Computer analysis of bacterial genome databases identified the presence of orthologues of the acnA gene (encodes aconitase A) in a number of putative prp operons. Homogeneous AcnA protein of S. enterica had strong aconitase activity and catalyzed the hydration of the 2-methyl-cis-aconitate to yield 2-methylisocitrate. The purification of this enzyme allows the complete reconstitution of the 2-methylcitric acid cycle in vitro using homogeneous preparations of the PrpE, PrpC, PrpD, AcnA, and PrpB enzymes. However, inactivation of the acnA gene did not block growth of S. enterica on propionate as carbon and energy source. The existence of a redundant aconitase activity (encoded by acnB) was postulated to be responsible for the lack of a phenotype in acnA mutant strains. Consistent with this hypothesis, homogeneous AcnB protein of S. enterica also had strong aconitase activity and catalyzed the conversion of 2-methyl-cis-aconitate into 2-methylisocitrate. To address the involvement of AcnB in propionate catabolism, an acnA and acnB double mutant was constructed, and this mutant strain cannot grow on propionate even when supplemented with glutamate. The phenotype of this double mutant indicates that the aconitase enzymes are required for the 2-methylcitric acid cycle during propionate catabolism.     

Studies of propionate toxicity in Salmonella enterica identify 2-methylcitrate as a potent inhibitor of cell growth

Salmonella enterica serovar Typhimurium LT2 showed increased sensitivity to propionate when the 2-methylcitric acid cycle was blocked. A derivative of a prpC mutant (which lacked 2-methylcitrate synthase activity) resistant to propionate was isolated, and the mutation responsible for the newly acquired resistance to propionate was mapped to the citrate synthase (gltA) gene. These results suggested that citrate synthase activity was the source of the increased sensitivity to propionate observed in the absence of the 2-methylcitric acid cycle. DNA sequencing of the wild-type and mutant gltA alleles revealed that the ATG start codon of the wild-type gene was converted to the rare GTG start codon in the revertant strain. This result suggested that lower levels of this enzyme were present in the mutant. Consistent with this change, cell-free extracts of the propionate-resistant strain contained 12-fold less citrate synthase activity. This was interpreted to mean that, in the wild-type strain, high levels of citrate synthase activity were the source of a toxic metabolite. In vitro experiments performed with homogeneous citrate synthase enzyme indicated that this enzyme was capable of synthesizing 2-methylcitrate from propionyl-CoA and oxaloacetate. This result lent further support to the in vivo data, which suggested that citrate synthase was the source of a toxic metabolite.     

2-Methylcitrate Dehydratase,a New Enzyme Functioning at the Methylcitric Acid Cycle of Propionate Metabolism

2-Methylcitrate dehydratase (2-methylcitrate hydro-lyase), a new enzyme functioning at the methylcitric acid cycle of propionyl-CoA oxidation, was present in the cell-free extract of Yarrowia (Saccharomycopsis) lipolytica. The enzyme was separated from the usual aconitate hydratase (EC 4.2.1.3) of the yeast with DEAE-Sephadex A-50 column chromatography. The enzyme was able to catalyze a reversible reaction between 2-methylcitrate and 2-methyl-cis-aconitate, but showed no activity on threo-d_s-2-methylisocitrate, citrate, cis- or trans-aconitate, threo-d_s-, threo-DL- or erythro-l_s-isocitrate, DL-homocitrate or other hydroxy-acids tested.

In contrast, the other enzyme fraction separated as aconitate hydratase by chromatography showed no activity on synthetic 2-methylcitrate, but was able to catalyze strongly a reversible reaction between 2-methyl-cis-aconitate and threo-d_s-2-methylisocitrate.

From these findings, the previously proposed cycle sequence was revised at the following broken arrows: propionyl-CoA+oxaloacetate → (CoASH+) 2-methylcitrate ? 2-methyl-cis-aconitate ? threo-d_s-2-methylisocitrate → pyruvate+succinate (→→oxaloacetate).

2-Methylcitrate dehydratase showed maximum activity at pH 6.5 to 7.0 and at 25 to 40°C. The enzyme was stable at temperatures up to 40°C and at pH 6.5 to 7.5, but labile in Tris-HCl buffer. The synthesis of this enzyme was constitutive in this yeast, although it was slightly repressed by glucose.     

Investigation of central carbon metabolism and the 2-methylcitrate cycle in Corynebacterium glutamicum by metabolic profiling using gas chromatography-mass spectrometry

The 2-methylcitrate cycle as the primary way to metabolize propionate was investigated using metabolic profiling. For this purpose, a fast harvesting procedure was applied in which cells growing in liquid minimal medium were harvested by a short centrifugation and freeze-dried. Subsequently, gas chromatography–mass spectrometry of polar extracts derivatized by MSTFA was employed for metabolite characterization. Routinely more than 300 different peaks were obtained in the chromatograms, and 74 substances were identified unequivocally by using pure standards. The procedure provided reliable data which closely relate to prior knowledge on flux distributions during growth on glucose and acetate as carbon sources.

Propionate degradation via the 2-methylcitrate cycle was demonstrated on the metabolite level by the detection of the intermediates 2-methylcitrate and 2-methylisocitrate. Further characterization of the 2-methylcitrate cycle was carried out by comparing different mutant strains of this pathway. The growth deficit of a prpD2-mutant strain observed when propionate is added to a culture growing on acetate indicates that the toxic effect of propionate is based on the accumulation of 2-methylcitrate. It could also be shown that the 2-methylcitrate cycle is active in the absence of propionate and might fulfill house-keeping functions in the degradation of fatty acids or branched-chain amino acids.     

Identification of the 2-methylcitrate pathway involved in the catabolism of propionate in the polyhydroxyalkanoate-producing strain Burkholderia sacchari IPT101(T) and analysis of a mutant accumulating a copolyester with higher 3-hydroxyvalerate content.

Burkholderia sacchari IPT101(T) induced the formation of 2-methylcitrate synthase and 2-methylisocitrate lyase when it was cultivated in the presence of propionic acid. The prp locus of B. sacchari IPT101(T) is required for utilization of propionic acid as a sole carbon source and is relevant for incorporation of 3-hydroxyvalerate (3HV) into copolyesters, and it was cloned and sequenced. Five genes (prpR, prpB, prpC, acnM, and ORF5) exhibited identity to genes located in the prp loci of other gram-negative bacteria. prpC encodes a 2-methylcitrate synthase with a calculated molecular mass of 42,691 Da. prpB encodes a 2-methylisocitrate lyase. The levels of PrpC and PrpB activity were much lower in propionate-negative mutant IPT189 obtained from IPT101(T) and were heterologously expressed in Escherichia coli. The acnM gene (ORF4) and ORF5, which are required for conversion of 2-methylcitric acid to 2-methylisocitric acid in Ralstonia eutropha HF39, are also located in the prp locus. The translational product of ORF1 (prpR) had a calculated molecular mass of 70,598 Da and is a putative regulator of the prp cluster. Three additional open reading frames (ORF6, ORF7, and ORF8) whose functions are not known were located adjacent to ORF5 in the prp locus of B. sacchari, and these open reading frames have not been found in any other prp operon yet. In summary, the organization of the prp genes of B. sacchari is similar but not identical to the organization of these genes in other bacteria investigated recently. In addition, this study provided a rationale for the previously shown increased molar contents of 3HV in copolyesters accumulated by a B. sacchari mutant since it was revealed in this study that the mutant is defective in prpC.     

Novel Inhibitors of Cholesterol Degradation in Mycobacterium tuberculosis Reveal How the Bacterium’s Metabolism Is Constrained by the Intracellular Environment

Mycobacterium tuberculosis (Mtb) relies on a specialized set of metabolic pathways to support growth in macrophages. By conducting an extensive, unbiased chemical screen to identify small molecules that inhibit Mtb metabolism within macrophages, we identified a significant number of novel compounds that limit Mtb growth in macrophages and in medium containing cholesterol as the principle carbon source. Based on this observation, we developed a chemical-rescue strategy to identify compounds that target metabolic enzymes involved in cholesterol metabolism. This approach identified two compounds that inhibit the HsaAB enzyme complex, which is required for complete degradation of the cholesterol A/B rings. The strategy also identified an inhibitor of PrpC, the 2-methylcitrate synthase, which is required for assimilation of cholesterol-derived propionyl-CoA into the TCA cycle. These chemical probes represent new classes of inhibitors with novel modes of action, and target metabolic pathways required to support growth of Mtb in its host cell. The screen also revealed a structurally-diverse set of compounds that target additional stage(s) of cholesterol utilization. Mutants resistant to this class of compounds are defective in the bacterial adenylate cyclase Rv1625/Cya. These data implicate cyclic-AMP (cAMP) in regulating cholesterol utilization in Mtb, and are consistent with published reports indicating that propionate metabolism is regulated by cAMP levels. Intriguingly, reversal of the cholesterol-dependent growth inhibition caused by this subset of compounds could be achieved by supplementing the media with acetate, but not with glucose, indicating that Mtb is subject to a unique form of metabolic constraint induced by the presence of cholesterol.     

细菌中2-甲基柠檬酸循环研究进展

2-甲基柠檬酸循环广泛分布于细菌中,参与丙酸或丙酰-CoA的分解代谢。我们一直致力于微生物代谢调控方面的研究,并以苏云金芽胞杆菌为研究对象在2-甲基柠檬酸循环的代谢调控及生理功能方面取得了新的进展。本文将从2-甲基柠檬酸循环关键酶基因的组成、关键酶基因的转录调控和该循环参与的生理功能3个方面介绍细菌中2-甲基柠檬酸循环的研究进展。同时,对该循环研究中存在的相关科学问题和未来的研究重点作简要评述,并对该循环关键酶作为药物靶标在病原菌感染防治方面的应用进行展望。     

Dual role of isocitrate lyase 1 in the glyoxylate and methylcitrate cycles in Mycobacterium tuberculosis

The role of isocitrate lyase (ICL) in the glyoxylate cycle and its necessity for persistence and virulence of Mycobacterium tuberculosis has been well described. Recent reports have alluded to an additional role for this enzyme in M. tuberculosis metabolism, specifically for growth on propionate. A product of beta-oxidation of odd-chain fatty acids is propionyl-CoA. Clearance of propionyl-CoA and the by-products of its metabolism via the methylcitrate cycle is vital due to their potentially toxic effects. Although the genome of M. tuberculosis encodes orthologues of two of the three enzymes of the methylcitrate cycle, methylcitrate synthase and methylcitrate dehydratase, it does not appear to contain a distinct 2-methylisocitrate lyase (MCL). Detailed structural analysis of the MCL from Escherichia coli suggested that the differences in substrate specificity between MCLs and ICLs could be attributed to three conserved amino acid substitutions in the active site, suggesting an MCL signature. However, here we provide enzymatic evidence that shows that despite the absence of the MCL signature, ICL1 from M. tuberculosis can clearly function as a MCL. Furthermore, the crystal structure of ICL1 with pyruvate and succinate bound demonstrates that the active site can accommodate the additional methyl group without significant changes to the structure.     

Identification of two prpDBC gene clusters in Corynebacterium glutamicum and their involvement in propionate degradation via the 2-methylcitrate cycle

Genome sequencing revealed that the Corynebacterium glutamicum genome contained, besides gltA, two additional citrate synthase homologous genes (prpC) located in two different prpDBC gene clusters, which were designated prpD1B1C1 and prpD2B2C2. The coding regions of the two gene clusters as well as the predicted gene products showed sequence identities of about 70 to 80%. Significant sequence similarities were found also to the prpBCDE operons of Escherichia coli and Salmonella enterica, which are known to encode enzymes of the propionate-degrading 2-methylcitrate pathway. Homologous and heterologous overexpression of the C. glutamicum prpC1 and prpC2 genes revealed that their gene products were active as citrate synthases and 2-methylcitrate synthases. Growth tests showed that C. glutamicum used propionate as a single or partial carbon source, although the beginning of the exponential growth phase was strongly delayed by propionate for up to 7 days. Compared to growth on acetate, the specific 2-methylcitrate synthase activity increased about 50-fold when propionate was provided as the sole carbon source, suggesting that in C. glutamicum the oxidation of propionate to pyruvate occurred via the 2-methylcitrate pathway. Additionally, two-dimensional gel electrophoresis experiments combined with mass spectrometry showed strong induction of the expression of the C. glutamicum prpD2B2C2 genes by propionate as an additional carbon source. Mutational analyses revealed that only the prpD2B2C2 genes were essential for the growth of C. glutamicum on propionate as a sole carbon source, while the function of the prpD1B1C1 genes remains obscure.     

Metabolic engineering of strains of Ralstonia eutropha and Pseudomonas putida for biotechnological production of 2-methylcitric acid

In this study strains of Ralstonia eutropha H16 and Pseudomonas putida KT2440 were engineered which are suitable for biotechnological production of 2-methylcitric acid (2MC). Analysis of a previous mutant of R. eutropha able to accumulate 2MC recommended this strain as a candidate for fermentative production of 2MC. This knowledge was used for construction of strains of R. eutropha H16 and P. putida KT2440 capable of enhanced production of 2MC. In both bacteria the chromosomal genes encoding the 2-methyl-cis-aconitate hydratase (acnM) were disrupted by directed insertion of a copy of an additional 2-methylcitrate synthase gene (prpC) yielding strains R. eutropha DeltaacnM(Re)OmegaKmprpC(Pp) and P. putida DeltaacnM(Pp)OmegaKmprpC(Re). In both strains 2-methylcitrate synthase was expressed under control of the constitutive kanamycin-resistance gene (OmegaKm) resulting in up to 20-fold higher specific 2-methylcitrate synthase activities in comparison to the wild type. The disruption of the acnM gene by insertion of prpC led to a propionate- and levulinate-negative phenotype of the engineered strains, and analysis of supernatant of these strains revealed overproduction and accumulation of 2MC in the medium. A two stage cultivation regime comprising an exponential growth phase and a 2MC production phase was developed and applied to both engineered strains for optimum production of 2MC. Whereas gluconate, fructose or succinate were provided as carbon source for the exponential growth phase, a combination of propionate or levulinate as precursor substrate for provision of propionyl-CoA and succinate or fumarate as precursor substrate for provision of oxaloacetate were used in the production phase to make sure that the 2-methylcitrate synthase was provided with their substrates. Employing the optimised feeding regime P. putida DeltaacnM(Pp)OmegaKmprpC(Re) and R. eutropha DeltaacnM(Re)OmegaKmprpC(Pp) produced 2MC up to maximal concentrations of 7.2 g/L or 26.5 mM and 19.2 g/L or 70.5 mM, respectively, during 144 h of cultivation.     

Characterization of the propionyl-CoA synthetase (PrpE) enzyme of Salmonella enterica: residue Lys592 is required for propionyl-AMP synthesis

The propionyl-CoA synthetase (PrpE) enzyme of Salmonella enterica catalyzes the first step of propionate catabolism, i.e., the activation of propionate to propionyl-CoA. The PrpE enzyme was purified, and its kinetic properties were determined. Evidence is presented that the conversion of propionate to propionyl-CoA proceeds via a propionyl-AMP intermediate. Kinetic experiments demonstrated that propionate was the preferred acyl substrate (kcat/Km = 1644 mM(-1) x s(-1)). Adenosine 5'-propyl phosphate was a potent inhibitor of the enzyme, and inhibition kinetics identified a Bi Uni Uni Bi Ping Pong mechanism for the reaction catalyzed by the PrpE enzyme. Site-directed mutagenesis was used to change the primary sequence of the wild-type protein at positions G245A, P247A, K248A, K248E, G249A, K592A, and K592E. Mutant PrpE proteins were purified, and the effects of the mutations on enzyme activity were investigated. Both PrpEK592 mutant proteins (K592A and K592E) failed to convert propionate to propionyl-CoA, and plasmids containing these alleles of prpE failed to restore growth on propionate of S. enterica carrying null prpE alleles on their chromosome. Both PrpEK592 mutant proteins converted propionyl-AMP to propionyl-CoA, suggesting residue K592 played no discernible role in thioester bond formation. To the best of our knowledge, these mutant proteins are the first acyl-CoA synthetases reported that are defective in adenylation activity.     

Purification and properties of the Streptomyces peucetius DpsC beta-ketoacyl:acyl carrier protein synthase III that specifies the propionate-starter unit for type II polyketide biosynthesis.

Biosynthesis of the polyketide-derived carbon skeleton of daunorubicin (DNR) begins with propionate rather than acetate, which is the starter unit for most other aromatic polyketides. The dpsCgene has been implicated in specifying the unique propionate-starter unit, and it encodes a protein that is very similar to the Escherichia coli beta-ketoacyl:acyl carrier protein (ACP) synthase III (FabH or KS III) enzyme of fatty acid biosynthesis. Purified DpsC was found to use propionyl-coenzyme A as substrate and to be acylated by propionate at the Ser-118 residue. DpsC exhibits KS III activity in catalyzing the condensation of propionyl-CoA and malonyl-ACP, and also functions as an acyltransferase in the transfer of propionate to an ACP. The DpsC enzyme has a high-substrate specificity, utilizing only propionyl-CoA, and not malonyl-CoA, 2-methylmalonyl-CoA or acetyl-CoA, as the starter unit of DNR biosynthesis.     

In Salmonella enterica, 2-Methylcitrate Blocks Gluconeogenesis

Strains of Salmonella enterica serovar Typhimurium LT2 lacking a functional 2-methylcitric acid cycle (2-MCC) display increased sensitivity to propionate. Previous work from our group indicated that this sensitivity to propionate is in part due to the production of 2-methylcitrate (2-MC) by the Krebs cycle enzyme citrate synthase (GltA). Here we report in vivo and in vitro data which show that a target of the 2-MC isomer produced by GltA (2-MC^GltA) is fructose-1,6-bisphosphatase (FBPase), a key enzyme in gluconeogenesis. Lack of growth due to inhibition of FBPase by 2-MC^GltA was overcome by increasing the level of FBPase or by micromolar amounts of glucose in the medium. We isolated an fbp allele encoding a single amino acid substitution in FBPase (S123F), which allowed a strain lacking a functional 2-MCC to grow in the presence of propionate. We show that the 2-MC^GltA and the 2-MC isomer synthesized by the 2-MC synthase (PrpC; 2-MC^PrpC) are not equally toxic to the cell, with 2-MC^GltA being significantly more toxic than 2-MC^PrpC. This difference in 2-MC toxicity is likely due to the fact that as a si-citrate synthase, GltA may produce multiple isomers of 2-MC, which we propose are not substrates for the 2-MC dehydratase (PrpD) enzyme, accumulate inside the cell, and have deleterious effects on FBPase activity. Our findings may help explain human inborn errors in propionate metabolism.Humans have used fermentation as an effective method of preservation for a wide variety of foods (41). Today, the weak short-chain fatty acids (SCFAs) produced by fermentation, such as acetic, propionic, butyric, and lactic acids, are widely used as food preservatives and in pre- and postharvest agricultural processes (34, 38, 45). Propionate, one of the most abundant SCFAs found in the environment (12), is widely used as a preservative of baked goods in the food industry (38).While SCFAs such as propionate are extensively used as food preservatives, our understanding of how microbial growth is prevented by them is incomplete. Early studies argued that growth inhibition either was caused by dissipation of the proton motive force (4, 48) or was due to decreases in intracellular pH (15, 48) or the intracellular accumulation of the propionate anion (46, 47). More recently, the global affects of SCFAs on gene expression (1, 43, 44) and protein synthesis (8, 37, 52, 56) were reported, revealing wide-ranging effects on gene expression in response to propionate in the environment (43). Evidence also suggests that central metabolic processes may be inhibited by SCFAs or their catabolites. An overview of the effects of propionate on the cell can be seen in Fig. ​Fig.11.Open in a separate windowFIG. 1.Overview of propionate metabolism and toxicity in Salmonella.Propionyl coenzyme A (Pr-CoA), an intermediate in propionate metabolism, was shown to inhibit pyruvate dehydrogenase in Rhodobacter sphaeroides (40) and Aspergillus niger (10) and competitively inhibit citrate synthase in Escherichia coli (39). 2-Methylcitrate (2-MC), the product of the condensation of oxaloacetate (OAA) and Pr-CoA, was shown to inhibit growth of Salmonella enterica, but the mechanism of action remained unclear (28) (Fig. ​(Fig.1).1). With such broad negative effects exerted by propionate or its catabolites, the best strategy for microbes to deal with SCFAs such as propionate is to efficiently catabolize them into central metabolites (Fig. ​(Fig.11).S. enterica, like many other enteric bacteria, is exposed to high levels of propionate in human digestive tracts with total SCFA levels varying from 20 to 300 mM and propionate reaching levels as high as 23.1 mmol/kg (9, 17). To cope with such high concentrations of propionate, this bacterium and other enterobacteria like E. coli utilize the 2-methylcitric acid cycle (2-MCC) to convert propionate to pyruvate (31, 53). In S. enterica, the prpBCDE operon encodes most of the 2-MCC enzymes (30). These genes encode a 2-methylisocitrate lyase (PrpB), a 2-methylcitrate synthase (PrpC), a 2-methylcitrate dehydratase (PrpD), and a propionyl coenzyme A (CoA) synthetase (PrpE) (Fig. ​(Fig.1).1). Early work with S. enterica showed that insertion elements placed within the prpBCDE operon greatly increased the sensitivity of S. enterica to propionate (23). Strains carrying insertions in prpE, however, were still able to grow on propionate and were not sensitive to propionate because acetyl-CoA synthetase (Acs) compensates for the lack of PrpE (32).The goal of the studies reported here was to identify a target of 2-MC in S. enterica. Our in vivo and in vitro data support the conclusion that 2-MC inhibits fructose-1,6-bisphosphatase (FBPase), a key enzyme of gluconeogenesis. The inhibition of FBPase blocks the synthesis of glucose, with the concomitant broad negative effects on cell function. We show that while both the 2-MC synthase (PrpC) and citrate synthase (GltA) enzymes synthesize 2-MC, the 2-MC made by GltA (2-MC^GltA) is more toxic to the cell than the 2-MC made by PrpC (2-MC^PrpC), and we suggest that the reason for this toxicity is due to the difference in stereochemistry of the GltA and PrpC reaction products.     

A degradation pathway of propionate in Salmonella typhimurium LT-2

Salmonella typhimurium LT-2 can utilize propionate as its sole carbon source. Studies on growth, oxidation by resting cell suspensions and by permeabilized cells, suggest that the propionate is transported by the acetate system. This result was confirmed using labeled propionate and acetate. ATP-monocarboxylate phosphotransferase, acyl-CoA orthophosphate acyl-transferase, propionyl-CoA dehydrogenase, acrylyl-CoA hydratase, lactate dehydrogenase, phosphoenolpyruvate (PEP) synthase and PEP-carboxylase activities have been identified in extracts of cells grown on propionate. Mutants deficient in PEP-carboxylase and synthase are unable to utilize propionate. On the basis of results obtained, it seems that the propionate degradation pathway occurs via acrylate and that PEP-synthase and PEP-carboxylase are essential enzymes.     

The three-dimensional crystal structure of the PrpF protein of Shewanella oneidensis complexed with trans-aconitate: insights into its biological function

In bacteria, the dehydration of 2-methylcitrate to yield 2-methylaconitate in the 2-methylcitric acid cycle is catalyzed by a cofactor-less (PrpD) enzyme or by an aconitase-like (AcnD) enzyme. Bacteria that use AcnD also require the function of the PrpF protein, whose function was previously unknown. To gain insights into the function of PrpF, the three-dimensional crystal structure of the PrpF protein from the bacterium Shewanella oneidensis was solved at 2.0 A resolution. The protein fold of PrpF is strikingly similar to those of the non-PLP-dependent diaminopimelate epimerase from Haemophilus influenzae, a putative proline racemase from Brucella melitensis, and to a recently deposited structure of a hypothetical protein from Pseudomonas aeruginosa. Results from in vitro studies show that PrpF isomerizes trans-aconitate to cis-aconitate. It is proposed that PrpF catalysis of the cis-trans isomerization proceeds through a base-catalyzed proton abstraction coupled with a rotation about C2-C3 bond of 2-methylaconitate, and that residue Lys73 is critical for PrpF function. The newly identified function of PrpF as a non-PLP-dependent isomerase, together with the fact that PrpD-containing bacteria do not require PrpF, suggest that the isomer of 2-methylaconitate that serves as a substrate of aconitase must have the same stereochemistry as that synthesized by PrpD. From this, it follows that the 2-methylaconitate isomer generated by AcnD is not a substrate of aconitase, and that PrpF is required to generate the correct isomer. As a consequence, the isomerase activity of PrpF may now be viewed as an integral part of the 2-methylcitric acid cycle.