Progress 10/01/02 to 09/30/06
Outputs
The TcuA protein has FAD-dependent tricarballylate dehydrogenase activity. Cloning and overexpression of tcuA. The tcuA+ gene was cloned and TcuA protein was isolated to >95% homogeneity. TcuA protein was yellow; UV-visible spectroscopy and mass spectrometry experiments determined that FAD was the cofactor bound to TcuA. Results of HPLC gel permeation experiments indicate that TcuA acts as a monomer. Oxidation of tricarballylate by TcuA yields cis-aconitate. In vitro conditions were established for assaying the putative tricarballylate dehydrogenase activity of TcuA. Homogeneous TcuA was incubated with tricarballylate, and the composition of the reaction mixture was analyzed by HPLC. The identity of an enzyme-dependent compound eluting 8 min post injection was determined by mass spectrometry to be cis-aconitate. The affinity of TcuA for tricarballylate (Km = 3.8 mM), the maximal velocity of the reaction (Vmax = 7.9 mM min-1), the turnover number (kcat = 6.7 s-1), and
the catalytic efficiency of the enzyme (kcat/Km = 17.8 M-1 s-1) were determined spectrophotometrically. These are kinetic parameters that we predict will change in assays that include TcuB. Under the conditions used, TcuA activity was optimal at pH 7.5, and fastest rate of product formation was observed at 30C. TcuB contains Fe-S centers and heme. The C-terminal two thirds of the TcuB protein was predicted to be an integral cell membrane domain, while the N-terminal third was predicted to be cytosolic. We cloned and overexpressed the 5' half of the tcuB gene of S. enterica fused to a chitin-binding domain. After purification by affinity chromatography, the resulting peptide (~12 kDa) had the expected brown color and UV-visible spectrum observed with Fe-S containing proteins. We also cloned the entire tcuB gene, which was predicted to encode a 42-kDa protein. Trypsin treatment of cell membrane material from cell overexpressing tcuB+ gene resulted in a deeply red solution with the
characteristic spectrum of heme. Overproduction conditions were optimized by the addition of Fe to the medium and performing the experiments inside an anoxic chamber to avoid oxygen damage. TcuB of 95% purity has been obtained using midl detergents. Sufficient protein has been isolated to perform EPR experiments to identify the type of Fe/S centers in TucB. The presence of heme has been unequivocally established by spectroscopic means. Experiments aimed at establishing the redox potential of heme are in progress. The role of TcuB as an electron shuttle has been established, and we have preliminary evidence that quinones are likely the electron acceptor to which TcuB donates electron extracted from tricarballylate. Results from two-hybrid interaction experiments between TcuA and the cytosolic fragment of TcuB indicate that these two proteins do interact. Future experiments will dissect these interactions. The kinetic parameters of TcuA have been reassessed. Under the conditions tested
a >2 order of magnitude increase in the velocity of the reaction has been measured.
Impacts
We continue to advance our understanding of how prokaryotes use tricarballylate as carbon and energy source. It appears that there is only on enzyme involved, that is the FAD-dependent tricarballylate dehydrogenase (TcuA) enzyme. The strategy used by the cell to oxidize FADH2 back to FAD+ is simple but very efficient. The cell takes advantage of the fact that flavin nucleotides (ie, FMN, FAD) can perform one-electron chemistry through their semiquinone states. Hence, the system appears to have evolved an electron shuttle protein that would use one-electron carries such as Fe/S centers and heme to deliver the electrons from FADH2 to the pool of quinones in the inner membrane. This is a very clever strategy because by doing so, the cell can supplement the proton motive force that drives chemiosmotic ATP synthesis through the ATPase. The central premise of our working model is that tricarballylate is not a compound the cell wants to reach the cytosol because of its strong
inhibitory effect on aconitase, a central metabolic enzyme. We think it makes sense to have a membrane-associated complex that couples transport of tricarballylate to its oxidation to cis-aconitate, a central metabolite. Our model predicts interactions amongst all three members of the complex; interactions amongst TcuA,B,C are yet to be explored. A model like the one we propose may provide evidence of a general physiological strategy cells use to avoid toxic effects by compounds that are structurally similar to central metabolites.
Publications
- Lewis, J. A. & J. C. Escalante-Semerena. 2006. The FAD-dependent tricarballylate dehudrogenase (TcuA) enzyme of Salmonella enterica converts tricarballylate into cis-aconitate. J. Bacteriol. 188:5479-5486.
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Progress 01/01/05 to 12/31/05
Outputs
The TcuA protein has FAD-dependent tricarballylate dehydrogenase activity. Cloning of tcuA. The tcuA gene was amplified using the genome of the wild-type strain as template. After an intermediate cloning step, the tcuA+ allele was cloned into a plasmid that directed the synthesis of the TcuA protein with a chitin-binding domain (CBD) fused to its C terminus (TcuA-CBD). Overproduction of TcuA protein. TcuA-CBD protein was overproduced in a commercially available E. coli everexpression strain. TcuA-CBD protein was isolated to homogeneity after binding the fused protein to a chitin affinity chromatography resin and eluting untagged protein after treatment with dithiothreitol. The solution of untagged TcuA protein was yellow, suggestive of a complex of the enzyme with its putative flavin cofactor. FAD, not FMN, is the cofactor bound to TcuA. We extracted the yellow cofactor bound to homogeneous TcuA, and used UV-visible spectroscopy and mass spectrometry to determine the
identity of the cofactor. The data unequivocally support the conclusion that FAD, not FMN, is the cofactor bound to TcuA. TcuA oxidation of tricarballylate yields cis-aconitate. In vitro conditions were established for assaying the putative tricarballylate dehydrogenase activity of TcuA. Homogeneous TcuA was incubated with tricarballylate, and the composition of the reaction mixture was analyzed by HPLC. The identity of an enzyme-dependent compound eluting 8 min post injection was determined by mass spectrometry to be cis-aconitate. The affinity of TcuA for tricarballylate, the maximal velocity of the reaction, the turnover number, and the catalytic efficiency of the enzyme were determined spectrophotometrically. TcuB contains Fe-S centers and heme. The C-terminal two thirds of the TcuB protein was predicted to be an integral cell membrane domain, while the N-terminal third was predicted to be cytosolic. We cloned and overexpressed the 5' half of the tcuB gene of S. enterica fused to
a chitin-binding domain. After purification by affinity chromatography, the resulting peptide (12 kDa) had the expected brown color and UV-visble spectrum observed with Fe-S containing proteins. We also cloned the entire tcuB gene, which was predicted to encode a 42 kDa protein. Trypsin treatment of cell membrane material from cell overexpressing tcuB gene resulted in a deeply red solution with the characteristic spectrum of heme. Initial quantitation using empty vector as a negative control indicated a dramatic increase in the amount of heme present in the membrane. Model for the re-oxidation of the FAD cofactor of TcuA. We have proposed that the TcuB protein serves as an electron shuttle between FADH2 in TcuA and electron carriers present in the cell membrane. The flow of electrons is proposed to go from FADH2 to Fe-S to heme to quinone. We are attemting to isolate TcuB and trying to set up an in vitro assay system to investigate this hypothesis.
Impacts
We have substantially improved our understanding of the tricarballylate utilization pathway used by prokaryotes to generate carbon and energy to grow. The identification of the biochemistry underpinning the critical first step of the pathway (catalyzed by TcuA) provides important clues into the role of the TcuB protein. What we have learned about the TcuB protein provides a framework for testing the hypothesis that the role of this protein is to reoxidize the electron carrier present in TcuA, so more substrate can be oxidized. It appears that the cell has evolved a FADH2 reoxidation system that takes advantage of the electron transport system in the cell membrane. A chain of one-electron transfer systems (Fe-S clusters, heme) appear to couple tricarballylate oxidation to the generation of the proton motive force needed to drive chemiosmotic ATP synthesis. Our model system centers on preventing the toxic effects of tricarballylate, which in addition to being an
excellent magnesium ion chelator, is also a potent inhibitor of aconitase, a key enzyme of the tricarboxylic acid cycle. We hypothesize that the TcuA, TcuB, and TcuC proteins work as a complex, in such a way that tricarballylate per se never reaches the cytosol. With this model in mind, we are planning experiments to probe for protein:protein interactions. A model like the one we are entertaining may provide evidence of a simple physiological strategy to avoid toxic effects by compounds that are structurally similar to central metabolites.
Publications
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Progress 01/01/04 to 12/31/04
Outputs
Genetic analysis of the tcuRABC operon. We generated a collection of strains deficient in their ability to use tricarballylate as carbon and energy source. Sixteen mutations causing a single-amino acid change were identified by DNA sequencing to be almost equeally dispersed amongst the tcuRABC genes. In addition, two insertion elements were isolated, one in the tcuR gene, the other one in the tcuA gene. We performed in vivo experiments to demonstrate that tcuR and tcuABC are two separate transcriptional units, and that tcuABC in fact comprise an operon. Defining each function by complementation. We used plasmids encoding individual tcu functions to demonstrate that TcuC is responsible for the transport of tricarballylate across the inner membrane. We also used in vivo approaches to show that the TcuC protein can transport citrate in addition to tricarballylate. Complementation studies with several different plasmids confirmed that the tcuABC genes comprise an operon.
We showed that the tcuABC functions are necessary and sufficient to allow Escherichia coli, which normally cannot grow on tricarballylate, to do so, indicating that no other functions unique to Salmonella enterica are needed for the catabolism of tricarballylate. Hypothetical activities of the TcuA and TcuB proteins. Our bioinformatics analyses of the tcuA and tcuB genes and gene products led us to hypothesize that TcuA is likely a flavin-dependent ocidoreductase responsible for the conversion of tricarballylate to cis-aconitate, which can enter the TCA cycle as a substrate of aconitase. We also proposed that TcuB is an Fe-S-containing electrontransferase needed to shuttle electrons to the electron transport system of the bacterium. This hypothesis is based on the predicted association of the C-terminal domain of TcuB with the inner membrane. Experiments aimed at testing these hypotheses are under way.
Impacts
The identification of the genes encoding the functions needed for the catabolism of tricarballylate is an important step toward an understanding of the biochemistry underpinning this metabolic pathway. A better understanding of the enzymology of this process may lead to rational drug design to inhibit enzymes involved in this pathway, thus preventing grass tetany.
Publications
- Lewis, J. A., A. R. Horswill, B. Schwem, and J. C. Escalante-Semerena. 2004. The tricarballylate utilization (tcuRABC) genes of Salmonella enterica serovar Typhimurium LT2. J. Bacteriol. 186:1629-1637.
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Progress 01/01/03 to 12/31/03
Outputs
The genes of S. enterica serovar Typhimurium LT2 encoding functions needed for the utilization of tricarballylate as a carbon and energy source were identified and their location in the chromosome established. Three of the tricarballylate utilization genes are organized as an operon; a fourth gene, is located immediately upstream to the operon. The operon encoding tricarballylate-degrading enzymes and the gene encoding the protein that regulates the expression of the operon share the same direction of transcription but are independently transcribed. All the tricarballylate catabolic genes are missing in the Escherichia coli chromosome. The last gene of the operon is proposed to encode an integral membrane protein whose role is to transport tricarballylate across the cell membrane. The gene encoding the tricarballylate transporter was sufficient to allow E. coli K12 to grow on citrate (a tricarballylate analog), but did not allow growth of this bacterium on
tricarballylate. E. coli carrying a plasmid with wild-type alleles of the tricarballylate genes grew on tricarballylate, suggesting that these genes encoded the only functions unique to S. enterica that were needed to catabolize tricarballylate. Analyses of the predicted amino acid sequences of two of the tricarballylate-degrading enzymes is a flavoprotein, and TcuB is likely anchored to the cell membrane, and probably contains one or more iron, sulfur centers. Two of the enzymes are proposed to work in concert to oxidize tricarballylate to cis-aconitate, which is further catabolized via the Krebs cycle. The glyoxylate shunt is not required for growth of S. enterica on tricarballylate. A model for tricarballylate catabolism in S. enterica is proposed.
Impacts
The identification of the genes encoding the functions needed for the catabolism of tricarballylate is an important step toward an understanding of the biochemistry underpinning this metabolic pathway. A better understanding of the enzymology of this process may lead to rational drug design to inhibit enzymes involved in this pathway, thus preventing grass tetany.
Publications
- No publications reported this period
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Progress 01/01/02 to 12/31/02
Outputs
This year of funding has allowed us to define the organization of four genes whose functions are required for the catabolism of tricarballylate in the enterobacterium Salmonella enterica. Analysis of these genes suggests that two of the encoded proteins (TcuA, TcuB) are likely to have enzymatic activity, one (TcuC) may be responsible for the transport of tricarballylate into the cell from the environment, and the fourth gene appears to encode a protein (TcuR) needed to regulate the expression of the operon. Using a reporter system under the control of the promoter driving the expression of the tcuABC genes, we have established that: i) TcuR is needed for expression; ii) that expression is triggered by the presence of tricarballylate in the environment; and iii) that TcuR is a sensor/response regulator transducing the signal (presence of tricarballylate) that controls expression of the operon. Data indicate that the signal sensed is tricarballylate and not a catabolite
of it. Preliminary data suggest that the tcuR gene is not part of the operon, and that tcuR is an independently controlled gene. No data is available at this point regarding the control of tcuR gene expression. We have defined each gene by complementation analysis. Each gene has been cloned under the control of a promoter whose activity is responsive to the presence of arabinose in the medium. Consistent with other data, we have established four complementation groups, and the nature of the lesions in mutant strains representative of each complementation group was determined by DNA sequencing.
Impacts
High levels of tricarballylate in the rumen has been correlated to grass tetany, a disease characterized by a pronounced magnesium ion deficiency in ruminants. Identification of the genes and enzymes involved in tricarballylate catabolism will allow genetic engineering of microorganisms that are natural inhabitants of the rumen to be constructed with the capability of lowering the level of tricarballylate in their environment. The availability of genetically engineered organisms capable of catabolizing tricarballylate will provide a natural way of reducing (if not eliminating) the incidence of grass tetany in agriculturally relevant mammals.
Publications
- No publications reported this period
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