Different localization in rat brain of the novel cytosolic ketone body-utilizing enzyme, acetoacetyl-CoA synthetase, as compared to succinyl-CoA:3-oxoacid CoA-transferase. (33/146)

In lipogenic tissue cytosol, ketone bodies are known to be activated by acetoacetyl-CoA synthetase (AACS) and incorporated into cholesterol and fatty acids. In order to investigate the physiological role of AACS in the brain, we examined the localization of AACS mRNA in rat brain by in situ hybridization using a labeled probe. High labeling was observed in the midbrain, pons/medulla, cerebral cortex, hippocampus and cerebellum, and the localization profile of AACS mRNA was different from that of succinyl-CoA:3-oxoacid CoA-transferase (SCOT), a mitochondrial ketone body-activating enzyme. In addition, the expression of AACS mRNA in the cerebellum was restricted primarily to glial cells, while in the cerebral cortex, it was restricted to neuronal cells. Streptozotocin treatment caused remarkable decreases in AACS mRNA levels in all regions where expression was observed, but changes in SCOT mRNA levels were not observed. These results suggest that the physiological role of AACS is different from that of SCOT and varies depending upon its localization in the brain.  (+info)

Characterization of the genes encoding beta-ketoadipate: succinyl-coenzyme A transferase in Pseudomonas putida. (34/146)

beta-Ketoadipate:succinyl-coenzyme A transferase (beta-ketoadipate:succinyl-CoA transferase) (EC 2.8.3.6) carries out the penultimate step in the conversion of benzoate and 4-hydroxybenzoate to tricarboxylic acid cycle intermediates in bacteria utilizing the beta-ketoadipate pathway. This report describes the characterization of a DNA fragment from Pseudomonas putida that encodes this enzyme. The fragment complemented mutants defective in the synthesis of the CoA transferase, and two proteins of sizes appropriate to encode the two nonidentical subunits of the enzyme were produced in Escherichia coli when the fragment was placed under the control of a phage T7 promoter. DNA sequence analysis revealed two open reading frames, designated pcaI and pcaJ, that were separated by 8 bp, suggesting that they may comprise an operon. A comparison of the deduced amino acid sequence of the P. putida CoA transferase genes with the sequences of two other bacterial CoA transferases and that of succinyl-CoA:3-ketoacid CoA transferase from pig heart suggests that the homodimeric structure of the mammalian enzyme may have resulted from a gene fusion of the bacterial alpha and beta subunit genes during evolution. Conserved functional groups important to the catalytic activity of CoA transferases were also identified.  (+info)

Crystallographic trapping of the glutamyl-CoA thioester intermediate of family I CoA transferases. (35/146)

Coenzyme A transferases are involved in a broad range of biochemical processes in both prokaryotes and eukaryotes, and exhibit a diverse range of substrate specificities. The YdiF protein from Escherichia coli O157:H7 is an acyl-CoA transferase of unknown physiological function, and belongs to a large sequence family of CoA transferases, present in bacteria to humans, which utilize oxoacids as acceptors. In vitro measurements showed that YdiF displays enzymatic activity with short-chain acyl-CoAs. The crystal structures of YdiF and its complex with CoA, the first co-crystal structure for any Family I CoA transferase, have been determined and refined at 1.9 and 2.0 A resolution, respectively. YdiF is organized into tetramers, with each monomer having an open alpha/beta structure characteristic of Family I CoA transferases. Co-crystallization of YdiF with a variety of CoA thioesters in the absence of acceptor carboxylic acid resulted in trapping a covalent gamma-glutamyl-CoA thioester intermediate. The CoA binds within a well defined pocket at the N- and C-terminal domain interface, but makes contact only with the C-terminal domain. The structure of the YdiF complex provides a basis for understanding the different catalytic steps in the reaction of Family I CoA transferases.  (+info)

Reconstruction and regulation of the central catabolic pathway in the thermophilic propionate-oxidizing syntroph Pelotomaculum thermopropionicum. (36/146)

Obligate anaerobic bacteria fermenting volatile fatty acids in syntrophic association with methanogenic archaea share the intermediate bottleneck step in organic-matter decomposition. These organisms (called syntrophs) are biologically significant in terms of their growth at the thermodynamic limit and are considered to be the ideal model to address bioenergetic concepts. We conducted genomic and proteomic analyses of the thermophilic propionate-oxidizing syntroph Pelotomaculum thermopropionicum to obtain the genetic basis for its central catabolic pathway. Draft sequencing and subsequent targeted gap closing identified all genes necessary for reconstructing its propionate-oxidizing pathway (i.e., methylmalonyl coenzyme A pathway). Characteristics of this pathway include the following. (i) The initial two steps are linked to later steps via transferases. (ii) Each of the last three steps can be catalyzed by two different types of enzymes. It was also revealed that many genes for the propionate-oxidizing pathway, except for those for propionate coenzyme A transferase and succinate dehydrogenase, were present in an operon-like cluster and accompanied by multiple promoter sequences and a putative gene for a transcriptional regulator. Proteomic analysis showed that enzymes in this pathway were up-regulated when grown on propionate; of these enzymes, regulation of fumarase was the most stringent. We discuss this tendency of expression regulation based on the genetic organization of the open reading frame cluster. Results suggest that fumarase is the central metabolic switch controlling the metabolic flow and energy conservation in this syntroph.  (+info)

A novel class of CoA-transferase involved in short-chain fatty acid metabolism in butyrate-producing human colonic bacteria. (37/146)

Bacterial butyryl-CoA CoA-transferase activity plays a key role in butyrate formation in the human colon, but the enzyme and corresponding gene responsible for this activity have not previously been identified. A novel CoA-transferase gene is described from the colonic bacterium Roseburia sp. A2-183, with similarity to acetyl-CoA hydrolase as well as 4-hydroxybutyrate CoA-transferase sequences. The gene product, overexpressed in an Escherichia coli lysate, showed activity with butyryl-CoA and to a lesser degree propionyl-CoA in the presence of acetate. Butyrate, propionate, isobutyrate and valerate competed with acetate as the co-substrate. Despite the sequence similarity to 4-hydroxybutyrate CoA-transferases, 4-hydroxybutyrate did not compete with acetate as the co-substrate. Thus the CoA-transferase preferentially uses butyryl-CoA as substrate. Similar genes were identified in other butyrate-producing human gut bacteria from clostridial clusters IV and XIVa, while other candidate CoA-transferases for butyrate formation could not be detected in Roseburia sp. A2-183. This suggests strongly that the newly identified group of CoA-transferases described here plays a key role in butyrate formation in the human colon.  (+info)

Transcriptional and functional analysis of oxalyl-coenzyme A (CoA) decarboxylase and formyl-CoA transferase genes from Lactobacillus acidophilus. (38/146)

Oxalic acid is found in dietary sources (such as coffee, tea, and chocolate) or is produced by the intestinal microflora from metabolic precursors, like ascorbic acid. In the human intestine, oxalate may combine with calcium, sodium, magnesium, or potassium to form less soluble salts, which can cause pathological disorders such as hyperoxaluria, urolithiasis, and renal failure in humans. In this study, an operon containing genes homologous to a formyl coenzyme A transferase gene (frc) and an oxalyl coenzyme A decarboxylase gene (oxc) was identified in the genome of the probiotic bacterium Lactobacillus acidophilus. Physiological analysis of a mutant harboring a deleted version of the frc gene confirmed that frc expression specifically improves survival in the presence of oxalic acid at pH 3.5 compared with the survival of the wild-type strain. Moreover, the frc mutant was unable to degrade oxalate. These genes, which have not previously been described in lactobacilli, appear to be responsible for oxalate degradation in this organism. Transcriptional analysis using cDNA microarrays and reverse transcription-quantitative PCR revealed that mildly acidic conditions were a prerequisite for frc and oxc transcription. As a consequence, oxalate-dependent induction of these genes occurred only in cells first adapted to subinhibitory concentrations of oxalate and then exposed to pH 5.5. Where genome information was available, other lactic acid bacteria were screened for frc and oxc genes. With the exception of Lactobacillus gasseri and Bifidobacterium lactis, none of the other strains harbored genes for oxalate utilization.  (+info)

Properties of succinyl-coenzyme A:L-malate coenzyme A transferase and its role in the autotrophic 3-hydroxypropionate cycle of Chloroflexus aurantiacus. (39/146)

The 3-hydroxypropionate cycle has been proposed to operate as the autotrophic CO2 fixation pathway in the phototrophic bacterium Chloroflexus aurantiacus. In this pathway, acetyl coenzyme A (acetyl-CoA) and two bicarbonate molecules are converted to malate. Acetyl-CoA is regenerated from malyl-CoA by L-malyl-CoA lyase. The enzyme forming malyl-CoA, succinyl-CoA:L-malate coenzyme A transferase, was purified. Based on the N-terminal amino acid sequence of its two subunits, the corresponding genes were identified on a gene cluster which also contains the gene for L-malyl-CoA lyase, the subsequent enzyme in the pathway. Both enzymes were severalfold up-regulated under autotrophic conditions, which is in line with their proposed function in CO2 fixation. The two CoA transferase genes were cloned and heterologously expressed in Escherichia coli, and the recombinant enzyme was purified and studied. Succinyl-CoA:L-malate CoA transferase forms a large (alphabeta)n complex consisting of 46- and 44-kDa subunits and catalyzes the reversible reaction succinyl-CoA + L-malate --> succinate + L-malyl-CoA. It is specific for succinyl-CoA as the CoA donor but accepts L-citramalate instead of L-malate as the CoA acceptor; the corresponding d-stereoisomers are not accepted. The enzyme is a member of the class III of the CoA transferase family. The demonstration of the missing CoA transferase closes the last gap in the proposed 3-hydroxypropionate cycle.  (+info)

Characterization of the beta-ketoadipate pathway in Sinorhizobium meliloti. (40/146)

Aromatic compounds represent an important source of energy for soil-dwelling organisms. The beta-ketoadipate pathway is a key metabolic pathway involved in the catabolism of the aromatic compounds protocatechuate and catechol, and here we show through enzymatic analysis and mutant analysis that genes required for growth and catabolism of protocatechuate in the soil-dwelling bacterium Sinorhizobium meliloti are organized on the pSymB megaplasmid in two transcriptional units designated pcaDCHGB and pcaIJF. The pcaD promoter was mapped by primer extension, and expression from this promoter is demonstrated to be regulated by the LysR-type protein PcaQ. Beta-ketoadipate succinyl-coenzyme A (CoA) transferase activity in S. meliloti was shown to be encoded by SMb20587 and SMb20588, and these genes have been renamed pcaI and pcaJ, respectively. These genes are organized in an operon with a putative beta-ketoadipyl-CoA thiolase gene (pcaF), and expression of the pcaIJF operon is shown to be regulated by an IclR-type transcriptional regulator, SMb20586, which we have named pcaR. We show that pcaR transcription is negatively autoregulated and that PcaR is a positive regulator of pcaIJF expression and is required for growth of S. meliloti on protocatechuate as the carbon source. The characterization of the protocatechuate catabolic pathway in S. meliloti offers an opportunity for comparison with related species, including Agrobacterium tumefaciens. Differences observed between S. meliloti and A. tumefaciens pcaIJ offer the first evidence of pca genes that may have been acquired after speciation in these closely related species.  (+info)