Screening, expression, purification and characterization of CoA-transferases for lactoyl-CoA generation.

Lactoyl-CoA is critical for the biosynthesis of biodegradable and biocompatible lactate-based copolymers, which have wide applications. However, reports on acetyl-CoA: lactate CoA-transferases (ALCTs) are rare. To exploit novel ALCTs, amino acid sequence similarity searches based on the CoA-transferases from Clostridium propionicum and Megasphaera elsdenii were conducted. Two known and three novel enzymes were expressed, purified and characterized. Three novel ALCTs were identified, one each from Megasphaera sp. DISK 18, Clostridium lactatifermentans An75 and Firmicutes bacterium CAG: 466. ME-PCT from Megasphaera elsdenii had the highest catalytic efficiency for both acetyl-CoA (264.22 s-1 mM-1) and D-lactate (84.18 s-1 mM-1) with a broad temperature range for activity and good stability. This study, therefore, offers novel and efficient enzymes for lactoyl-CoA generation. To our best knowledge, this is the first report on the systematic mining of ALCTs, which offers valuable new tools for the engineering of pathways that rely on these enzymes.

Identification and characterization of two bile acid coenzyme A transferases from Clostridium scindens, a bile acid 7α-dehydroxylating intestinal bacterium.

The human bile acid pool composition is composed of both primary bile acids (cholic acid and chenodeoxycholic acid) and secondary bile acids (deoxycholic acid and lithocholic acid). Secondary bile acids are formed by the 7α-dehydroxylation of primary bile acids carried out by intestinal anaerobic bacteria. We have previously described a multistep biochemical pathway in Clostridium scindens that is responsible for bile acid 7α-dehydroxylation. We have identified a large (12 kb) bile acid inducible (bai) operon in this bacterium that encodes eight genes involved in bile acid 7α-dehydroxylation. However, the function of the baiF gene product in this operon has not been elucidated. In the current study, we cloned and expressed the baiF gene in E. coli and discovered it has bile acid CoA transferase activity. In addition, we discovered a second bai operon encoding three genes. The baiK gene in this operon was expressed in E. coli and found to encode a second bile acid CoA transferase. Both bile acid CoA transferases were determined to be members of the type III family by amino acid sequence comparisons. Both bile acid CoA transferases had broad substrate specificity, except the baiK gene product, which failed to use lithocholyl-CoA as a CoA donor. Primary bile acids are ligated to CoA via an ATP-dependent mechanism during the initial steps of 7α-dehydroxylation. The bile acid CoA transferases conserve the thioester bond energy, saving the cell ATP molecules during bile acid 7α-dehydroxylation. ATP-dependent CoA ligation is likely quickly supplanted by ATP-independent CoA transfer.

Characterization of an acyl-CoA: carboxylate CoA-transferase from Aspergillus nidulans involved in propionyl-CoA detoxification.

Filamentous fungi metabolize toxic propionyl-CoA via the methylcitrate cycle. Disruption of the methylcitrate synthase gene leads to an accumulation of propionyl-CoA and attenuates virulence of Aspergillus fumigatus. However, addition of acetate, but not ethanol, to propionate-containing medium strongly reduces the accumulation of propionyl-CoA and restores growth of the methylcitrate synthase mutant. Therefore, the existence of a CoA-transferase was postulated, which transfers the CoASH moiety from propionyl-CoA to acetate and, thereby, detoxifying the cell. In this study, we purified the responsible protein from Aspergillus nidulans and characterized its biochemical properties. The enzyme used succinyl-, propionyl- and acetyl-CoA as CoASH donors and the corresponding acids as acceptor molecules. Although the protein displayed high sequence similarity to acetyl-CoA hydrolases this activity was hardly detectable. We additionally identified and deleted the coding DNA sequence of the CoA-transferase. The mutant displayed weak phenotypes in the presence of propionate and behaved like the wild type when no propionate was present. However, when a double-deletion mutant defective in both methylcitrate synthase and CoA-transferase was constructed, the resulting strain was unable to grow on media containing acetate and propionate as sole carbon sources, which confirmed the in vivo activity of the CoA-transferase.

In vitro synthesis of polyhydroxyalkanoates using thermostable acetyl-CoA synthetase, CoA transferase, and PHA synthase from thermotorelant bacteria.

Thermostable enzymes are required for the rapid and sustainable production of polyhydroxyalkanoate (PHA) in vitro. The in vitro synthesis of PHA using the engineered thermostable synthase PhaC1SG(STQK) has been reported; however, the non-thermostable enzymes acetyl-CoA synthetase (ACS) and CoA transferase (CT) from mesophilic strains were used as monomer-supplying enzymes in this system. In the present study, acs and ct were cloned from the thermophilic bacteria Pelotomaculum thermopropionicum JCM10971 and Thermus thermophilus JCM10941 to construct an in vitro PHA synthesis system using only thermostable enzymes. ACS from P. thermopropionicum (ACSPt) and CT from T. thermophilus (CTTt) were confirmed to have high thermostability, and their optimal temperatures were around 60°C and 75°C, respectively. The in vitro PHA synthesis was successfully performed by ACSPt, CTTt, PhaC1SG(STQK), and poly(3-hydroxybutyrate) [P(3HB)] was synthesized at 45°C. Furthermore, the yields of P(3HB) and P(lactate-co-3HB) at 37°C were 1.4-fold higher than those of the in vitro synthesis system with non-thermostable ACS and CT from mesophilic strains. Overall, the thermostable ACS and CT were demonstrated to be useful for the efficient in vitro PHA synthesis at relatively high temperatures.

Further purification and characterization of the succinyl-CoA:3-hydroxy-3-methylglutarate coenzyme A transferase from rat-liver mitochondria.

Succinyl-CoA:3-hydroxy-3-methylglutarate coenzyme A transferase, previously identified in rat-liver mitochondria (Deana et al. (1981), Biochim. Biophys. Acta 662, 119-124), was purified to near homogeneity and further characterized. After the last purification steps consisting of Ultrogel AcA-44 filtration and agarose-hexane-coenzyme A chromatography, the enzyme was apparently tetrameric with a mass of 48-52 kDa determined by gel filtration on Sephadex G-75, ultracentrifugation through a sucrose gradient and SDS-gel electrophoresis. By means of a HPLC technique developed for measuring the CoA esters we could determine the enzyme activity in both forward and reverse directions and show that the kinetic constants, i.e., Km of reactants and Vmax, are not too different for the two reactions. Double-reciprocal plots of the enzyme velocities versus the concentration of one substrate at different fixed concentrations of the other substrate gave families of straight lines converging below the substrate-abscissa for both forward and backward reactions, indicating a kinetic mechanism of rapid equilibrium random Bi-Bi type. The competitive inhibition of the product succinate with respect to both reactants, 3-hydroxy-3-methylglutarate and succinyl-CoA, as well as the Haldane relationships are consistent with this conclusion. An inhibitory effect on CoA transferase activity by acetate, acetoacetate, acetyl-CoA, acetoacetyl-CoA, coenzyme A, carnitine, ZnCl2 and high concentrations of the monovalent anions ClO4-, F-, I- and Cl- was also found.

Function and X-ray crystal structure of Escherichia coli YfdE.

Many food plants accumulate oxalate, which humans absorb but do not metabolize, leading to the formation of urinary stones. The commensal bacterium Oxalobacter formigenes consumes oxalate by converting it to oxalyl-CoA, which is decarboxylated by oxalyl-CoA decarboxylase (OXC). OXC and the class III CoA-transferase formyl-CoA:oxalate CoA-transferase (FCOCT) are widespread among bacteria, including many that have no apparent ability to degrade or to resist external oxalate. The EvgA acid response regulator activates transcription of the Escherichia coli yfdXWUVE operon encoding YfdW (FCOCT), YfdU (OXC), and YfdE, a class III CoA-transferase that is ~30% identical to YfdW. YfdW and YfdU are necessary and sufficient for oxalate-induced protection against a subsequent acid challenge; neither of the other genes has a known function. We report the purification, in vitro characterization, 2.1-Å crystal structure, and functional assignment of YfdE. YfdE and UctC, an orthologue from the obligate aerobe Acetobacter aceti, perform the reversible conversion of acetyl-CoA and oxalate to oxalyl-CoA and acetate. The annotation of YfdE as acetyl-CoA:oxalate CoA-transferase (ACOCT) expands the scope of metabolic pathways linked to oxalate catabolism and the oxalate-induced acid tolerance response. FCOCT and ACOCT active sites contain distinctive, conserved active site loops (the glycine-rich loop and the GNxH loop, respectively) that appear to encode substrate specificity.

Acetate:succinate CoA-transferase in the hydrogenosomes of Trichomonas vaginalis: identification and characterization.

Acetate:succinate CoA-transferases (ASCT) are acetate-producing enzymes in hydrogenosomes, anaerobically functioning mitochondria and in the aerobically functioning mitochondria of trypanosomatids. Although acetate is produced in the hydrogenosomes of a number of anaerobic microbial eukaryotes such as Trichomonas vaginalis, no acetate producing enzyme has ever been identified in these organelles. Acetate production is the last unidentified enzymatic reaction of hydrogenosomal carbohydrate metabolism. We identified a gene encoding an enzyme for acetate production in the genome of the hydrogenosome-containing protozoan parasite T. vaginalis. This gene shows high similarity to Saccharomyces cerevisiae acetyl-CoA hydrolase and Clostridium kluyveri succinyl-CoA:CoA-transferase. Here we demonstrate that this protein is expressed and is present in the hydrogenosomes where it functions as the T. vaginalis acetate:succinate CoA-transferase (TvASCT). Heterologous expression of TvASCT in CHO cells resulted in the expression of an active ASCT. Furthermore, homologous overexpression of the TvASCT gene in T. vaginalis resulted in an equivalent increase in ASCT activity. It was shown that the CoA transferase activity is succinate-dependent. These results demonstrate that this acetyl-CoA hydrolase/transferase homolog functions as the hydrogenosomal ASCT of T. vaginalis. This is the first hydrogenosomal acetate-producing enzyme to be identified. Interestingly, TvASCT does not share any similarity with the mitochondrial ASCT from Trypanosoma brucei, the only other eukaryotic succinate-dependent acetyl-CoA-transferase identified so far. The trichomonad enzyme clearly belongs to a distinct class of acetate:succinate CoA-transferases. Apparently, two completely different enzymes for succinate-dependent acetate production have evolved independently in ATP-generating organelles.

Formation of amyloid fibrils via longitudinal growth of oligomers.

Mature amyloid fibrils are believed to be formed by the lateral association of discrete structural units designated as protofibrils, but this lateral association of protofibrils has never been directly observed. We have recently characterized a thioesterase from Alcaligenes faecalis, which was shown to exist as homomeric oligomers with an average diameter of 21.6 nm consisting of 22 kDa subunits in predominantly beta-sheet structure. In this study, we have shown that upon incubation in a 75% ethanol solution, the oligomeric particles of protein were transformed into amyloid-like fibrils. TEM pictures obtained at various stages during fibril growth helped us to understand to a certain extent the early events in the fibrillization process. When incubated in 75% ethanol, oligomeric particles of protein grew to approximately 35-40 nm in diameter before fusion. Fusion of two oligomers of 35-40 nm resulted in the formation of a fibril. Fibril formation was accompanied by a reduction in the diameter of the particle to approximately 20-25 nm along with concomitant elongation to approximately 110 nm, indicating reorganization and strengthening of the structure. The elongation process continued by sequential addition of oligomeric units to give fibers 500-1000 nm in length with a further reduction in diameter to 17-20 nm. Further elongation resulted in the formation of fibers that were more than 4000 nm in length; the diameter, however, remained constant at 17-20 nm. These data clearly show that the mature fibrils have assembled via longitudinal growth of oligomers and not via lateral association of protofibrils.

New vectors for co-expression of proteins: structure of Bacillus subtilis ScoAB obtained by high-throughput protocols.

The Bacillus subtilis genes scoA and scoB encode subunits of the heteromeric enzyme ScoAB, a putative succinyl-CoA:acetoacetate coenzyme A transferase. High-throughput, ligation-independent cloning (LIC) vectors used extensively for production and purification of single proteins were modified to allow simultaneous expression of interacting proteins and selective purification of functional complexes. Transfer of the LIC region of vector pMCSG7 (L. Stols, M. Gu, L. Dieckman, R. Raffen, F.R. Collart, M.I. Donnelly. A new vector for high-throughput, ligation-independent cloning encoding a tobacco etch virus protease cleavage site. Protein Expr. Purif. (2002) 25, 8-15) into commercial vectors with alternative, compatible origins of replication allowed introduction of standard LIC PCR products into the vectors by uniform protocols. Replacement of the His-tag encoding region of pMCSG7 with a sequence encoding the S-tag enabled selective purification of interacting proteins based on the His-tag associated with one member of the complex. When expressed separately and mixed, the ScoAB subunits failed to interact productively; no transferase activity was detected, and S-tagged ScoB failed to co-purify with His-tagged ScoA. Co-expression, in contrast, generated active transferase that catalyzed the predicted reaction. The ScoAB complex was purified by standard high-throughput metal-ion affinity chromatography procedures, crystallized robotically, and its structure was determined by molecular replacement.

Identification of the cysteine residue exposed by the conformational change in pig heart succinyl-CoA:3-ketoacid coenzyme A transferase on binding coenzyme A.

Succinyl-CoA:3-ketoacid CoA transferase (SCOT) transfers CoA from succinyl-CoA to acetoacetate via a thioester intermediate with its active site glutamate residue, Glu 305. When CoA is linked to the enzyme, a cysteine residue can now be rapidly modified by 5,5'-dithiobis(2-nitrobenzoic acid), reflecting a conformational change of SCOT upon formation of the thioester. Since either Cys 28 or Cys 196 could be the target, each was mutated to Ser to distinguish between them. Like wild-type SCOT, the C196S mutant protein was modified rapidly in the presence of acyl-CoA substrates. In contrast, the C28S mutant protein was modified much more slowly under identical conditions, indicating that Cys 28 is the residue exposed on binding CoA. The specific activity of the C28S mutant protein was unexpectedly lower than that of wild-type SCOT. X-ray crystallography revealed that Ser adopts a different conformation than the native Cys. A chloride ion is bound to one of four active sites in the crystal structure of the C28S mutant protein, mimicking substrate, interacting with Lys 329, Asn 51, and Asn 52. On the basis of these results and the studies of the structurally similar CoA transferase from Escherichia coli, YdiF, bound to CoA, the conformational change in SCOT was deduced to be a domain rotation of 17 degrees coupled with movement of two loops: residues 321-329 that bury Cys 28 and interact with succinate or acetoacetate and residues 374-386 that interact with CoA. Modeling this conformational change has led to the proposal of a new mechanism for catalysis by SCOT.

Properties of succinyl-coenzyme A:D-citramalate coenzyme A transferase and its role in the autotrophic 3-hydroxypropionate cycle of Chloroflexus aurantiacus.

The phototrophic bacterium Chloroflexus aurantiacus uses the 3-hydroxypropionate cycle for autotrophic CO(2) fixation. This cycle starts with acetyl-coenzyme A (CoA) and produces glyoxylate. Glyoxylate is an unconventional cell carbon precursor that needs special enzymes for assimilation. Glyoxylate is combined with propionyl-CoA to beta-methylmalyl-CoA, which is converted to citramalate. Cell extracts catalyzed the succinyl-CoA-dependent conversion of citramalate to acetyl-CoA and pyruvate, the central cell carbon precursor. This reaction is due to the combined action of enzymes that were upregulated during autotrophic growth, a coenzyme A transferase with the use of succinyl-CoA as the CoA donor and a lyase cleaving citramalyl-CoA to acetyl-CoA and pyruvate. Genomic analysis identified a gene coding for a putative coenzyme A transferase. The gene was heterologously expressed in Escherichia coli and shown to code for succinyl-CoA:d-citramalate coenzyme A transferase. This enzyme, which catalyzes the reaction d-citramalate + succinyl-CoA --> d-citramalyl-CoA + succinate, was purified and studied. It belongs to class III of the coenzyme A transferase enzyme family, with an aspartate residue in the active site. The homodimeric enzyme composed of 44-kDa subunits was specific for succinyl-CoA as a CoA donor but also accepted d-malate and itaconate instead of d-citramalate. The CoA transferase gene is part of a cluster of genes which are cotranscribed, including the gene for d-citramalyl-CoA lyase. It is proposed that the CoA transferase and the lyase catalyze the last two steps in the glyoxylate assimilation route.

2-Methylbutyryl-CoA: succinate acyl-CoA transferase activity and function in Ascaris suum muscle.

A branched-chain acyl-CoA transferase activity which transfers coenzyme A from either 2-methylbutyryl or 2-methylvaleryl-CoA to succinate is present in the muscle mitochondria from the intestinal nematode, Ascaris suum. Its physiological function is discussed. This activity appears to differ from the previously described acetyl-CoA: propionate and propionyl-CoA:succinate acyl-CoA transferases on the basis of heat stability, substrate specificity and the requirement of a "factor" from boiled Ascaris mitochondria for optimal activity of only the branched-chain acyl-CoA transferase. The "factor" has been recovered from HPLC and some of its properties examined. It could not be replaced by a crude soluble fraction from rat liver mitochondria, or by adenine, guanine or inosine di- or triphosphates. Activity was lost upon ashing, but was not affected by treatment with either pepsin or chymotrypsin.

Coenzyme A transferase from Clostridium acetobutylicum ATCC 824 and its role in the uptake of acids.

Coenzyme A (CoA) transferase from Clostridium acetobutylicum ATCC 824 was purified 81-fold to homogeneity. This enzyme was stable in the presence of 0.5 M ammonium sulfate and 20% (vol/vol) glycerol, whereas activity was rapidly lost in the absence of these stabilizers. The kinetic binding mechanism was Ping Pong Bi Bi, and the Km values at pH 7.5 and 30 degrees C for acetate, propionate, and butyrate were, respectively, 1,200, 1,000, and 660 mM, while the Km value for acetoacetyl-CoA ranged from about 7 to 56 microM, depending on the acid substrate. The Km values for butyrate and acetate were high relative to the intracellular concentrations of these species; consequently, in vivo enzyme activity is expected to be sensitive to changes in those concentrations. In addition to the carboxylic acids listed above, this CoA transferase was able to convert valerate, isobutyrate, and crotonate; however, the conversion of formate, n-caproate, and isovalerate was not detected. The acetate and butyrate conversion reactions in vitro were inhibited by physiological levels of acetone and butanol, and this may be another factor in the in vivo regulation of enzyme activity. The optimum pH of acetate conversion was broad, with at least 80% of maximal activity from pH 5.9 to greater than 7.8. The purified enzyme was a heterotetramer with subunit molecular weights of about 23,000 and 25,000.

Purification and characterization of formyl-coenzyme A transferase from Oxalobacter formigenes.

Formyl-coenzyme A (formyl-CoA) transferase was purified from Oxalobacter formigenes by high-pressure liquid chromatography with hydrophobic interaction chromatography and by DEAE anion-exchange chromatography. The enzyme was a single entity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel permeation chromatography (Mr, 44,000). It had an isoelectric point of 4.7. The enzyme catalyzed the transfer of CoA from formyl-CoA to either oxalate or succinate. Apparent Km and Vmax values, respectively, were 3.0 mM and 29.6 mumols/min per mg for formyl-CoA with an excess of succinate. The maximum specific activity was 2.15 mumols of CoA transferred from formyl-CoA to oxalate per min per mg of protein.

Purification and properties of 4-hydroxybutyrate coenzyme A transferase from Clostridium aminobutyricum.

A new coenzyme A (CoA)-transferase from the anaerobe Clostridium aminobutyricum catalyzing the formation of 4-hydroxybutyryl-CoA from 4-hydroxybutyrate and acetyl-CoA is described. The enzyme was purified to homogeneity by standard techniques, including fast protein liquid chromatography under aerobic conditions. Its molecular mass was determined to be 110 kDa, and that of the only subunit was determined to be 54 kDa, indicating a homodimeric structure. Besides acetate and acetyl-CoA, the following substrates were detected (in order of decreasing kcat/Km): 4-hydroxybutyryl-CoA, butyryl-CoA and propionyl-CoA, vinyl-acetyl-CoA (3-butenoyl-CoA), and 5-hydroxyvaleryl-CoA. In an indirect assay the corresponding acids were also found to be substrates; however, DL-lactate, DL-2-hydroxybutyrate, DL-3-hydroxybutyrate, crotonate, and various dicarboxylates were not.

Propionate CoA-transferase from Clostridium propionicum. Cloning of gene and identification of glutamate 324 at the active site.

Propionate CoA-transferase from Clostridium propionicum has been purified and the gene encoding the enzyme has been cloned and sequenced. The enzyme was rapidly and irreversibly inactivated by sodium borohydride or hydroxylamine in the presence of propionyl-CoA. The reduction of the thiol ester between a catalytic site glutamate and CoA with borohydride and the cleavage by hydroxylamine were used to introduce a site-specific label, which was followed by MALDI-TOF-MS. This allowed the identification of glutamate 324 at the active site. Propionate CoA-transferase and similar proteins deduced from the genomes of Escherichia coli, Staphylococcus aureus, Bacillus halodurans and Aeropyrum pernix are proposed to form a novel subclass of CoA-transferases. Secondary structure element predictions were generated and compared to known crystal structures in the databases. A high degree of structural similarity was observed between the arrangement of secondary structure elements in these proteins and glutaconate CoA-transferase from Acidaminococcus fermentans.

Location of the two genes encoding glutaconate coenzyme A-transferase at the beginning of the hydroxyglutarate operon in Acidaminococcus fermentans.

Glutaconate coenzyme A-transferase (Gct) from Acidaminococcus fermentans consists of two subunits (GctA, 35725 Da and GctB, 29168 Da). The N-termini sequences of both subunits were determined. DNA sequencing of a subgenomic fragment of A. fermentans revealed that the genes encoding glutaconate CoA-transferase (gctAB) are located upstream of a gene cluster formed by gcdA, hgdC, hgdA and hgdB in this order. Further upstream of gctA, a DNA sequence was detected showing significant similarities to sigma 70-type promoters from Escherichia coli. Primer-extension analysis revealed that this specific DNA sequence was indeed the location of transcription initiation in A. fermentans. The entire gene cluster, 7.3 kb in length, comprising gctAB, gcdA and hgdCAB, has tentatively been named the hydroxyglutarate operon, since the enzymes encoded by these genes are involved in the conversion of (R)-2-hydroxyglutarate to crotonyl-CoA in the pathway of glutamate fermentation by A. fermentans. The genes gctAB were expressed together in E. coli. Cell-free extracts of a transformant E. coli strain contained glutaconate CoA-transferase at a specific activity of up to 30 U/mg protein. The recombinant enzyme was purified to homogeneity with a specific activity of 130 U/mg protein by ammonium sulfate fractionation and crystallisation. The amino acid residue directly involved in catalysis was tentatively identified as E54 of the small subunit of the enzyme (GctB).

Crystallization and preliminary crystallographic analysis of formyl-CoA tranferase from Oxalobacter formigenes.

Formyl-CoA transferase from Oxalobacter formigenes has been expressed as a recombinant protein in Escherichia coli and purified to homogeneity. Crystals of formyl-CoA transferase were grown at 293 K using polyethylene glycol 4000 as a precipitant. The diffraction pattern of flash-frozen crystals at 100 K extends to 2.2 A resolution with synchrotron radiation (lambda = 0.933 nm). The crystals are tetragonal and belong to space group I4, with unit-cell parameters a = b = 151.44, c = 99.49 A. The asymmetric unit contains one dimer and the solvent content is 53%. Formyl-CoA transferase was crystallized both as the apoenzyme and as its complex with coenzyme A.

Structure of the mammalian CoA transferase from pig heart.

Ketoacidosis affects patients who are deficient in the enzyme activity of succinyl-CoA:3-ketoacid CoA transferase (SCOT), since SCOT catalyses the activation of acetoacetate in the metabolism of ketone bodies. Thus far, structure/function analysis of the mammalian enzyme has been predicted based on the three-dimensional structure of a CoA transferase determined from an anaerobic bacterium that utilizes its enzyme for glutamate fermentation. To better interpret clinical data, we have determined the structure of a mammalian CoA transferase from pig heart by X-ray crystallography to 2.5 A resolution. Instrumental to the structure determination were selenomethionine substitution and the use of argon during purification and crystallization. Although pig heart SCOT adopts an alpha/beta protein fold, resembling the overall fold of the bacterial CoA transferase, several loops near the active site of pig heart SCOT follow different paths than the corresponding loops in the bacterial enzyme, accounting for differences in substrate specificities. Two missense mutations found associated with SCOT of ketoacidosis patients were mapped to a location in the structure that might disrupt the stabilization of the amino-terminal strand and thereby interfere with the proper folding of the protein into a functional enzyme.

Sequence of a cDNA clone encoding pig heart mitochondrial CoA transferase.

We have isolated a full-length cDNA clone encoding the cytoplasmic precursor to pig heart mitochondrial CoA transferase (succinyl-CoA:3-ketoacid coenzyme A transferase (3-oxoacid CoA transferase, EC 2.8.3.5], a key enzyme for ketone body catabolism. The deduced amino acid sequence indicates the presence of a 39-residue mitochondrial signal sequence and a 481-residue mature protein of molecular weight 52,197. CoA transferase is known to be susceptible to proteolytic cleavage to produce a nicked but active enzyme. We have identified the site of proteolysis, and analysis of the sequence in its vicinity suggests that the polypeptide may fold into two domains connected by a highly hydrophilic bridge.