CoA Donor Research Articles

Complete sequence randomness of lactate-based copolymers (LAHBs) with varied lactate monomer fractions employing a series of propionyl-CoA transferases

Previously, we biosynthesized an evolved version of a bio-based polylactide (PLA) on microbial platforms using our engineered lactate-polymerizing enzyme (LPE). This lactate (LA)-based copolyester, LAHB, has advantages over PLA, including improved flexibility and biodegradability, and its properties can be regulated through the LA fraction. To expand the LA-incorporation capacity and improve polymer properties, in the state of in vivo LAHB production, propionyl-CoA transferases (PCTs) that exhibited enhanced production of LA-CoA than the conventional PCTs were selected. Here, the present study has demonstrated that the LA fraction of LAHB could be altered using various PCTs. Enhanced PCT performance was achieved by balancing polymer production and cell growth. Both events are governed by the use of acetyl-CoA, a commonly shared key metabolite. This could be attributed to the different reactivities of individual PCTs towards acetyl-CoA, which serves both as a CoA donor and a leading compound in the TCA cycle. Interestingly, we found complete sequence randomness in the LAHB copolymers, independent of the LA fraction. The mechanism of LA fraction-independent sequence randomness is discussed. This new PCT-based strategy synergistically combines with the evolution of LPE to advance the LAHB project, and enables us to perform advanced applications other than LAHB production utilizing CoA-linked substrates.

Isophthalate:coenzyme A ligase initiates anaerobic degradation of xenobiotic isophthalate

BackgroundEnvironmental contamination from synthetic plastics and their additives is a widespread problem. Phthalate esters are a class of refractory synthetic organic compounds which are widely used in plastics, coatings, and for several industrial applications such as packaging, pharmaceuticals, and/or paints. They are released into the environment during production, use and disposal, and some of them are potential mutagens and carcinogens. Isophthalate (1,3-benzenedicarboxylic acid) is a synthetic chemical that is globally produced at a million-ton scale for industrial applications and is considered a priority pollutant. Here we describe the biochemical characterization of an enzyme involved in anaerobic degradation of isophthalate by the syntrophically fermenting bacterium Syntrophorhabdus aromaticivorans strain UI that activate isophthalate to isophthalyl-CoA followed by its decarboxylation to benzoyl-CoA.ResultsIsophthalate:Coenzyme A ligase (IPCL, AMP-forming) that activates isophthalate to isophthalyl-CoA was heterologously expressed in E. coli (49.6 kDa) for biochemical characterization. IPCL is homologous to phenylacetate-CoA ligase that belongs to the family of ligases that form carbon-sulfur bonds. In the presence of coenzyme A, Mg2+ and ATP, IPCL converts isophthalate to isophthalyl-CoA, AMP and pyrophosphate (PPi). The enzyme was specifically induced after anaerobic growth of S. aromaticivorans in a medium containing isophthalate as the sole carbon source. Therefore, IPCL exhibited high substrate specificity and affinity towards isophthalate. Only substrates that are structurally related to isophthalate, such as glutarate and 3-hydroxybenzoate, could be partially converted to the respective coenzyme A esters. Notably, no activity could be measured with substrates such as phthalate, terephthalate and benzoate. Acetyl-CoA or succinyl-CoA did not serve as CoA donors. The enzyme has a theoretical pI of 6.8 and exhibited optimal activity between pH 7.0 to 7.5. The optimal temperature was between 25 °C and 37 °C. Denaturation temperature (Tm) of IPCL was found to be at about 63 °C. The apparent KM values for isophthalate, CoA, and ATP were 409 μM, 642 μM, and 3580 μM, respectively. Although S. aromaticivorans is a strictly anaerobic bacterium, the enzyme was found to be oxygen-insensitive and catalysed isophthalyl-CoA formation under both anoxic and oxic conditions.ConclusionWe have successfully cloned the ipcl gene, expressed and characterized the corresponding IPCL enzyme, which plays a key role in isophthalate activation that initiates its activation and further degradation by S. aromaticivorans. Its biochemical characterization represents an important step in the elucidation of the complete degradation pathway of isophthalate.

Enzymes involved in the anaerobic degradation of ortho-phthalate by the nitrate-reducing bacterium Azoarcus sp. strain PA01.

The pathway of anaerobic degradation of o-phthalate was studied in the nitrate-reducing bacterium Azoarcus sp. strain PA01. Differential two-dimensional protein gel profiling allowed the identification of specifically induced proteins in o-phthalate-grown compared to benzoate-grown cells. The genes encoding o-phthalate-induced proteins were found in a 9.9 kb gene cluster in the genome of Azoarcus sp. strain PA01. The o-phthalate-induced gene cluster codes for proteins homologous to a dicarboxylic acid transporter, putative CoA-transferases and a UbiD-like decarboxylase that were assigned to be specifically involved in the initial steps of anaerobic o-phthalate degradation. We propose that o-phthalate is first activated to o-phthalyl-CoA by a putative succinyl-CoA-dependent succinyl-CoA:o-phthalate CoA-transferase, and o-phthalyl-CoA is subsequently decarboxylated to benzoyl-CoA by a putative o-phthalyl-CoA decarboxylase. Results from in vitro enzyme assays with cell-free extracts of o-phthalate-grown cells demonstrated the formation of o-phthalyl-CoA from o-phthalate and succinyl-CoA as CoA donor, and its subsequent decarboxylation to benzoyl-CoA. The putative succinyl-CoA:o-phthalate CoA-transferase showed high substrate specificity for o-phthalate and did not accept isophthalate, terephthalate or 3-fluoro-o-phthalate whereas the putative o-phthalyl-CoA decarboxylase converted fluoro-o-phthalyl-CoA to fluoro-benzoyl-CoA. No decarboxylase activity was observed with isophthalyl-CoA or terephthalyl-CoA. Both enzyme activities were oxygen-insensitive and inducible only after growth with o-phthalate. Further degradation of benzoyl-CoA proceeds analogous to the well-established anaerobic benzoyl-CoA degradation pathway of nitrate-reducing bacteria.

Identification of an itaconic acid degrading pathway in itaconic acid producing Aspergillus terreus.

Itaconic acid, one of the most promising and flexible bio-based chemicals, is mainly produced by Aspergillus terreus. Previous studies to improve itaconic acid production in A. terreus through metabolic engineering were mainly focused on its biosynthesis pathway, while the itaconic acid-degrading pathway has largely been ignored. In this study, we used transcriptomic, proteomic, bioinformatic, and in vitro enzymatic analyses to identify three key enzymes, itaconyl-CoA transferase (IctA), itaconyl-CoA hydratase (IchA), and citramalyl-CoA lyase (CclA), that are involved in the catabolic pathway of itaconic acid in A. terreus. In the itaconic acid catabolic pathway in A. terreus, itaconic acid is first converted by IctA into itaconyl-CoA with succinyl-CoA as the CoA donor, and then itaconyl-CoA is hydrated into citramalyl-CoA by IchA. Finally, citramalyl-CoA is cleaved into acetyl-CoA and pyruvate by CclA. Moreover, IctA can also catalyze the reaction between citramalyl-CoA and succinate to generate succinyl-CoA and citramalate. These results, for the first time, identify the three key enzymes, IctA, IchA, and CclA, involved in the itaconic acid degrading pathway in itaconic acid producing A. terreus. The results will facilitate the improvement of itaconic acid production by metabolically engineering the catabolic pathway of itaconic acid in A. terreus.

Fermentative production of enantiomerically pure S-1,2-propanediol from glucose by engineered E. coli strain.

The pure stereoisomers of 1,2-propanediol (1,2-PDO) could be used as starting materials to synthesize high value-added specialty chemicals and chiral pharmaceutical products. As the stereoisomers of 1,2-PDO cannot be obtained by traditional chemical synthesis processes, biotechnological processes have gained increasing attention. However, to our knowledge, the production of S-1,2-PDO directly from glucose has not been previously reported. In this study, we demonstrate a novel artificial pathway to convert L-lactic acid to S-1,2-PDO and its integration into the genome of Escherichia coli strain BW25113∆poxB with synchronous deletion of genes responsible for branch metabolic pathways from glucose. L-lactate production was increased by replacing the native D-lactate dehydrogenase with the L-lactate dehydrogenase from Bacillus coagulans. The methylglyoxal bypass pathway was blocked to avoid synthesis of a racemic mixture of D- and L-lactate and prevent the accumulation of methylglyoxal, a toxic intermediate. To further improve the yield of S-1,2-PDO, a novel cofactor regeneration system was introduced by combining pyruvate decarboxylase and acetaldehyde-CoA dehydrogenase II to simultaneously regenerate NADH and the CoA donor of acetyl-CoA for the lactate conversion pathway. Finally, 13.7mM S-1,2-PDO with >99% enantiomeric purity was directly produced from glucose by disrupting the major carbon-competing pathways and strengthening the lactate transformation pathway. This study demonstrates the first attempt to synthesize S-1,2-PDO by direct fermentation of glucose.

Improved production of poly(lactic acid)-like polyester based on metabolite analysis to address the rate-limiting step.

The biosynthesis of poly(lactic acid) (PLA)-like polymers, composed of >99 mol% lactate and a trace amount of 3-hydroxybutyrate, in engineered Corynebacterium glutamicum consists of two steps; the generation of the monomer substrate lactyl-coenzyme A (CoA) and the polyhydroxyalkanoate (PHA) synthase-catalyzed polymerization of lactyl-CoA. In order to increase polymer productivity, we explored the rate-limiting step in PLA-like polymer synthesis based on quantitative metabolite analysis using liquid chromatography mass spectroscopy (LC-MS). A significant pool of lactyl-CoA was found during polymer synthesis. This result suggested that the rate-limitation occurred at the polymerization step. Accordingly, the expression level of PHA synthase was increased by means of codon-optimization of the corresponding gene that consequently led to an increase in polymer content by 4.4-fold compared to the control. Notably, the codon-optimization did not significantly affect the concentration of lactyl-CoA, suggesting that the polymerization reaction was still the rate-limiting step upon the overexpression of PHA synthase. Another important finding was that the generation of lactyl-CoA was concomitant with a decrease in the acetyl-CoA level, indicating that acetyl-CoA served as a CoA donor for lactyl-CoA synthesis. These results show that obtaining information on the metabolite concentrations is highly useful for improving PLA-like polymer production. This strategy should be applicable to a wide range of PHA-producing systems.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-014-0083-2) contains supplementary material, which is available to authorized users.

Characterization of propionate CoA-transferase from Ralstonia eutropha H16

In this study, a propionate CoA-transferase (H16_A2718; EC 2.8.3.1) from Ralstonia eutropha H16 (Pct(Re)) was characterized in detail. Glu342 was identified as catalytically active amino acid residue via site-directed mutagenesis. Activity of Pct(Re) was irreversibly lost after the treatment with NaBH₄ in the presence of acetyl-CoA as it is shown for all CoA-transferases from class I, thereby confirming the formation of the covalent enzyme-CoA intermediate by Pct(Re). In addition to already known CoA acceptors for Pct Re such as 3-hydroxypropionate, 3-hydroxybutyrate, acrylate, succinate, lactate, butyrate, crotonate and 4-hydroxybutyrate, it was found that glycolate, chloropropionate, acetoacetate, valerate, trans-2,3-pentenoate, isovalerate, hexanoate, octanoate and trans-2,3-octenoate formed also corresponding CoA-thioesters after incubation with acetyl-CoA and Pct(Re). Isobutyrate was found to be preferentially used as CoA acceptor amongst other carboxylates tested in this study. In contrast, no products were detected with acetyl-CoA and formiate, bromopropionate, glycine, pyruvate, 2-hydroxybutyrate, malonate, fumarate, itaconate, β-alanine, γ-aminobutyrate, levulate, glutarate or adipate as potential CoA acceptor. Amongst CoA donors, butyryl-CoA, crotonyl-CoA, 3-hydroxybutyryl-CoA, isobutyryl-CoA, succinyl-CoA and valeryl-CoA apart from already known propionyl-CoA and acetyl-CoA could also donate CoA to acetate. The highest rate of the reaction was observed with 3-hydroxybutyryl-CoA (2.5 μmol mg⁻¹ min⁻¹). K(m) values for propionyl-CoA, acetyl-CoA, acetate and 3-hydroxybutyrate were 0.3, 0.6, 4.5 and 4.3 mM, respectively. The rather broad substrate range might be a good starting point for enzyme engineering approaches and for the application of Pct(Re) in biotechnological polyester production.

Caffeate Respiration in the Acetogenic Bacterium Acetobacterium woodii: a Coenzyme A Loop Saves Energy for Caffeate Activation

The anaerobic acetogenic bacterium Acetobacterium woodii couples reduction of caffeate with electrons derived from molecular hydrogen to the synthesis of ATP by a chemiosmotic mechanism with sodium ions as coupling ions. Caffeate is activated to caffeyl coenzyme A (caffeyl-CoA) prior to its reduction, and the caffeate reduction operon encodes an ATP-dependent caffeyl-CoA synthetase that is thought to catalyze the initial caffeate activation. The operon also encodes a potential CoA transferase, the product of carA, which was thought to be involved in subsequent ATP-independent caffeate activation. To prove the proposed function of carA, we overproduced its protein in Escherichia coli and then purified it. Purified CarA drives the formation of caffeyl-CoA from caffeate with hydrocaffeyl-CoA as the CoA donor. The dependence of the reaction on caffeate and hydrocaffeyl-CoA followed Michaelis-Menten kinetics, with apparent K(m) values of 75 ± 5 μM for caffeate and 8 ± 2 μM for hydrocaffeyl-CoA. The enzyme activity had broad ranges of pH and temperature optima. In addition to being able to use caffeate, CarA could use p-coumarate and ferulate but not cinnamate, sinapate, or p-hydroxybenzoate as a CoA acceptor. Neither acetyl-CoA nor butyryl-CoA served as the CoA donor for CarA. The enzyme uses a ping-pong mechanism for CoA transfer and is the first classified member of a new subclass of family I CoA transferases that has two catalytic domains on one polypeptide chain. Apparently, CarA catalyzes an energy-saving CoA loop for caffeate activation in the steady state of caffeate respiration.

A propionate CoA-transferase of Ralstonia eutropha H16 with broad substrate specificity catalyzing the CoA thioester formation of various carboxylic acids

In this study, we have investigated a propionate CoA-transferase (Pct) homologue encoded in the genome of Ralstonia eutropha H16. The corresponding gene has been cloned into the vector pET-19b to yield a histidine-tagged enzyme which was expressed in Escherichia coli BL21 (DE3). After purification, high-performance liquid chromatography/mass spectrometry (HPLC/MS) analyses revealed that the enzyme exhibits a broad substrate specificity for carboxylic acids. The formation of the corresponding CoA-thioesters of acetate using propionyl-CoA as CoA donor, and of propionate, butyrate, 3-hydroxybutyrate, 3-hydroxypropionate, crotonate, acrylate, lactate, succinate and 4-hydroxybutyrate using acetyl-CoA as CoA donor could be shown. According to the substrate specificity, the enzyme can be allocated in the family I of CoA-transferases. The apparent molecular masses as determined by gel filtration and detected by SDS polyacrylamide gel electrophoresis were 228 and 64kDa, respectively, and point to a quaternary structure of the native enzyme (α4). The enzyme exhibited similarities in sequence and structure to the well investigated Pct of Clostridium propionicum. It does not contain the typical conserved (S)ENG motif, but the derived motif sequence EXG with glutamate 342 to be, most likely, the catalytic residue. Due to the homo-oligomeric structure and the sequence differences with the subclasses IA-C of family I CoA-transferases, a fourth subclass of family I is proposed, comprising - amongst others - the Pcts of R. eutropha H16 and C. propionicum. A markerless precise-deletion mutant R. eutropha H16∆pct was generated. The growth and accumulation behaviour of this mutant on gluconate, gluconate plus 3,3'-dithiodipropionic acid (DTDP), acetate and propionate was investigated but resulted in no observable phenotype. Both, the wild type and the mutant showed the same growth and storage behaviour with these carbon sources. It is probable that R. eutropha H16 is upregulating other CoA-transferase(s) or CoA-synthetase(s), thereby compensating for the lacking Pct. The ability of R. eutropha H16 to substitute absent enzymes by isoenzymes has been already shown in different other studies in the past.

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.

Substrate Recognition by β-Ketoacyl-ACP Synthases

β-Ketoacyl-ACP synthase (KAS) enzymes catalyze Claisen condensation reactions in the fatty acid biosynthesis pathway. These reactions follow a ping-pong mechanism in which a donor substrate acylates the active site cysteine residue after which the acyl group is condensed with the malonyl-ACP acceptor substrate to form a β-ketoacyl-ACP. In the priming KASIII enzymes the donor substrate is an acyl-CoA while in the elongating KASI and KASII enzymes the donor is an acyl-ACP. Although the KASIII enzyme in Escherichia coli (ecFabH) is essential, the corresponding enzyme in Mycobacterium tuberculosis (mtFabH) is not, suggesting that the KASI or II enzyme in M. tuberculosis (KasA or KasB, respectively) must be able to accept a CoA donor substrate. Since KasA is essential, the substrate specificity of this KASI enzyme has been explored using substrates based on phosphopantetheine, CoA, ACP, and AcpM peptide mimics. This analysis has been extended to the KASI and KASII enzymes from E. coli (ecFabB and ecFabF) where we show that a 14-residue malonyl-phosphopantetheine peptide can efficiently replace malonyl-ecACP as the acceptor substrate in the ecFabF reaction. While ecFabF is able to catalyze the condensation reaction when CoA is the carrier for both substrates, the KASI enzymes ecFabB and KasA have an absolute requirement for an ACP substrate as the acyl donor. Provided that this requirement is met, variation in the acceptor carrier substrate has little impact on the k(cat)/K(m) for the KASI reaction. For the KASI enzymes we propose that the binding of ecACP (AcpM) results in a conformational change that leads to an open form of the enzyme to which the malonyl acceptor substrate binds. Finally, the substrate inhibition observed when palmitoyl-CoA is the donor substrate for the KasA reaction has implications for the importance of mtFabH in the mycobacterial FASII pathway.

Point Mutations (Q19P and N23K) Increase the Operational Solubility of a 2α-O-Benzoyltransferase that Conveys Various Acyl Groups from CoA to a Taxane Acceptor

Two site-directed mutations within the wild-type 2-O-benzoyltransferase (tbt) cDNA, from Taxus cuspidata plants, yielded an encoded protein containing replacement amino acids at Q19P and N23K that map to a solvent-exposed loop region. The likely significant changes in the biophysical properties invoked by these mutations caused the overexpressed, modified TBT (mTBT) to partition into the soluble enzyme fraction about 5-fold greater than the wild-type enzyme. Sufficient protein could now be acquired to examine the scope of the substrate specificity of mTBT by incubation with 7,13-O,O-diacetyl-2-O-debenzoylbaccatin III that was mixed individually with various substituted benzoyls, alkanoyls, and (E)-butenoyl CoA donors. The mTBT catalyzed the conversion of each 7,13-O,O-diacetyl-2-O-debenzoylbaccatin III to several 7,13-O,O-diacetyl-2-O-acyl-2-O-debenzoylbaccatin III analogues. The relative catalytic efficiency of mTBT with the 7,13-O,O-diacetyl-2-O-debenzoyl surrogate substrate and heterole carbonyl CoA substrates was slightly greater than with the natural aroyl substrate benzoyl CoA, while substituted benzoyl CoA thioesters were less productive. Short-chain hydrocarbon carbonyl and cyclohexanoyl CoA thioesters were also productive, where C(4) substrates were transferred by mTBT with approximately 10- to 17-fold greater catalytic efficiency compared to the transfer of benzoyl. The described broad specificity of mTBT suggests that a plethora of 2-O-acyl variants of the antimitotic paclitaxel can be assembled through biocatalytic sequences.

An N-Aroyltransferase of the BAHD Superfamily Has Broad Aroyl CoA Specificity in Vitro with Analogues of N-Dearoylpaclitaxel

The native N-debenzoyl-2'-deoxypaclitaxel:N-benzoyltransferase (NDTBT), from Taxus plants, transfers a benzoyl group from the corresponding CoA thioester to the amino group of the beta-phenylalanine side chain of N-debenzoyl-2'-deoxypaclitaxel, which is purportedly on the paclitaxel (Taxol) biosynthetic pathway. To elucidate the substrate specificity of NDTBT overexpressed in Escherichia coli, the purified enzyme was incubated with semisynthetically derived N-debenzoyltaxoid substrates and aroyl CoA donors (benzoyl; ortho-, meta-, and para-substituted benzoyls; various heterole carbonyls; alkanoyls; and butenoyl), which were obtained from commercial sources or synthesized via a mixed anhydride method. Several unnatural N-aroyl-N-debenzoyl-2'-deoxypaclitaxel analogues were biocatalytically assembled with catalytic efficiencies (V(max)/K(M)) ranging between 0.15 and 1.74 nmol.min(-1).mM(-1). In addition, several N-acyl-N-debenzoylpaclitaxel variants were biosynthesized when N-debenzoylpaclitaxel and N-de(tert-butoxycarbonyl)docetaxel (i.e., 10-deacetyl-N-debenzoylpaclitaxel) were used as substrates. The relative velocity (v(rel)) for NDTBT with the latter two N-debenzoyl taxane substrates ranged between approximately 1% and 200% for the array of aroyl CoAs compared to benzoyl CoA. Interestingly, NDTBT transferred hexanoyl, acetyl, and butyryl more rapidly than butenoyl or benzoyl from the CoA donor to taxanes with isoserinoyl side chains, whereas N-debenzoyl-2'-deoxypaclitaxel was more rapidly converted to its N-benzoyl derivative than to its N-alkanoyl or N-butenoyl congeners. Biocatalytic N-acyl transfer of novel acyl groups to the amino functional group of N-debenzoylpaclitaxel and its 2'-deoxy precursor reveal the surprisingly indiscriminate specificity of this transferase. This feature of NDTBT potentially provides a tool for alternative biocatalytic N-aroylation/alkanoylation to construct next generation taxanes or other novel bioactive diterpene compounds.

Pathway identification combining metabolic flux and functional genomics analyses: Acetate and propionate activation by Corynebacterium glutamicum

Corynebacterium glutamicum can utilize acetic acid and propionic acid for growth and amino acid production. Growth on acetate as sole carbon source requires acetate activation by acetate kinase (AK) and phosphotransacetylase (PTA), encoded in the pta-ack operon. Genetic and enzymatic studies showed that these enzymes also catalyze propionate activation and were required for growth on propionate as sole carbon source. However, when glucose was present as a co-substrate strain lacking the AK-PTA pathway was still able to utilize acetate or propionate for growth indicating that an alternative activation pathway exists. As shown by 13C-labelling experiments, the carbon skeleton of acetate is conserved during activation to acetyl-CoA in this pathway. Metabolic flux analysis during growth on an acetate–glucose mixture revealed that in the absence of the AK-PTA pathway carbon fluxes in glycolysis, the tricarboxylic acid (TCA) cycle and anaplerosis via PEP carboxylase and/or pyruvate carboxylase were increased, while the glyoxylate cycle flux was decreased. DNA microarray experiments identified cg2840 as a constitutively and highly expressed gene putatively encoding a CoA transferase. Purified His-tagged Cg2840 protein was active as CoA transferase interconverting acetyl-, propionyl- and succinyl-moieties as CoA acceptors and donors. Strains lacking both the CoA transferase and the AK-PTA pathway could neither activate acetate nor propionate in the presence or absence of glucose. Thus, when these short-chain fatty acids are co-metabolized with other carbon sources, CoA transferase and the AK-PTA pathway constitute a redundant system for activation of acetate and propionate.

Measurement of the Energy-Generating Capacity of Human Muscle Mitochondria: Diagnostic Procedure and Application to Human Pathology

Diagnosis of mitochondrial disorders usually requires a muscle biopsy to examine mitochondrial function. We describe our diagnostic procedure and results for 29 patients with mitochondrial disorders. Muscle biopsies were from 43 healthy individuals and 29 patients with defects in one of the oxidative phosphorylation (OXPHOS) complexes, the pyruvate dehydrogenase complex (PDHc), or the adenine nucleotide translocator (ANT). Homogenized muscle samples were used to determine the oxidation rates of radiolabeled pyruvate, malate, and succinate in the absence or presence of various acetyl Co-A donors and acceptors, as well as specific inhibitors of tricarboxylic acid cycle or OXPHOS enzymes. We determined the rate of ATP production from oxidation of pyruvate. Each defect in the energy-generating system produced a specific combination of substrate oxidation impairments. PDHc deficiencies decreased substrate oxidation reactions containing pyruvate. Defects in complexes I, III, and IV decreased oxidation of pyruvate plus malate, with normal to mildly diminished oxidation of pyruvate plus carnitine. In complex V defects, pyruvate oxidation improved by addition of carbonyl cyanide 3-chlorophenyl hydrazone, whereas other oxidation rates were decreased. In most patients, ATP production was decreased. The proposed method can be successfully applied to the diagnosis of defects in PDHc, OXPHOS complexes, and ANT.

Properties of Succinyl-Coenzyme A: l -Malate Coenzyme A Transferase and Its Role in the Autotrophic 3-Hydroxypropionate Cycle of Chloroflexus aurantiacus

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.

Crystallographic Trapping of the Glutamyl-CoA Thioester Intermediate of Family I CoA Transferases

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.

Defects in Cell Growth Regulation by C18:0-Ceramide and Longevity Assurance Gene 1 in Human Head and Neck Squamous Cell Carcinomas

In this study, endogenous long chain ceramides were measured in 32 human head and neck squamous cell carcinoma (HNSCC) and 10 nonsquamous head and neck carcinoma tumor tissues, as compared with adjacent noncancerous tissues, by liquid chromatography/mass spectroscopy. Interestingly, only one specific ceramide, C(18:0)-ceramide, was selectively down-regulated in the majority of HNSCC tumor tissues. On the other hand, in nonsquamous tumor tissues, this selectivity for C18-ceramide was not detected. These data suggested the hypotheses that decreased levels of C18-ceramide might impart a growth advantage to HNSCC cells and that increased generation of C18-ceramide may be involved in the inhibition of growth. These roles were examined by reconstitution of C18-ceramide at physiologically relevant concentrations in UM-SCC-22A cells (squamous cell carcinoma of hypopharynx) via overexpression of mammalian upstream regulator of growth and differentiation factor 1 (mUOG1), a mouse homologue of longevity assurance gene 1 (mLAG1), which has been shown to specifically induce the generation of C18-ceramide. Liquid chromatography/mass spectroscopy analysis showed that overexpression of the mLAG1/mUOG1 resulted in increased levels of only C(18:0)-ceramide by approximately 2-fold, i.e. concentrations similar to those of normal head and neck tissues. Importantly, increased generation of C18-ceramide by mLAG1/mUOG1 inhibited cell growth (approximately 70-80%), which mechanistically involved the modulation of telomerase activity and induction of apoptotic cell death by mitochondrial dysfunction. In conclusion, this study demonstrates, for the first time, a biological role for LAG1 and C18-ceramide in the regulation of growth of HNSCC.

Characterization of a novel CI-976-sensitive lysophospholipid acyltransferase that is associated with the Golgi complex

Recent studies have identified a novel lysophospholipid acyltransferase (LPAT) that is associated with the Golgi complex and that is sensitive to the previously characterized acyl–CoA cholesterol acyltransferase inhibitor, 2,2-methyl- N-(2,4,6-trimethoxyphenyl)dodecanamide (CI-976). Here we show that besides acting on exogenous lysophospholipid (LPL) substrates, the CI-976-sensitive LPAT is also capable of reacylating endogenous Golgi LPL substrates, preferentially lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE). Moreover, using exogenous substrates, we find that the CI-976-sensitive LPAT is capable of using a variety of fatty acyl–CoA donors ranging in chain length from 10 to 20 carbons. Additional characterization demonstrates that the CI-976-sensitive LPAT is ubiquitously expressed in rat tissues, is tightly associated with Golgi membranes, and has a pH optimum between pH 7.0 and 8.0. These studies further define a unique LPC/LPE-specific LPAT from Golgi membranes that likely has a novel function in membrane trafficking.

Involvement of coenzyme A esters and two new enzymes, an enoyl-CoA hydratase and a CoA-transferase, in the hydration of crotonobetaine to L-carnitine by Escherichia coli.

Two proteins (CaiB and CaiD) were found to catalyze the reversible biotransformation of crotonobetaine to L-carnitine in Escherichia coli in the presence of a cosubstrate (e.g., gamma-butyrobetainyl-CoA or crotonobetainyl-CoA). CaiB (45 kDa) and CaiD (27 kDa) were purified in two steps to electrophoretic homogeneity from overexpression strains. CaiB was identified as crotonobetainyl-CoA:carnitine CoA-transferase by MALDI-TOF mass spectrometry and enzymatic assays. The enzyme exhibits high cosubstrate specificity to CoA derivatives of trimethylammonium compounds. In particular, the N-terminus of CaiB shows significant identity with other CoA-transferases (e.g., FldA from Clostridium sporogenes, Frc from Oxalobacter formigenes, and BbsE from Thauera aromatica) and CoA-hydrolases (e.g., BaiF from Eubacterium sp.). CaiD was shown to be a crotonobetainyl-CoA hydratase using MALDI-TOF mass spectrometry and enzymatic assays. Besides crotonobetainyl-CoA CaiD is also able to hydrate crotonyl-CoA with a significantly lower Vmax (factor of 10(3)) but not crotonobetaine. The substrate specificity of CaiD and its homology to the crotonase confirm this enzyme as a new member of the crotonase superfamily. Concluding these results, it was verified that hydration of crotonobetaine to L-carnitine proceeds at the CoA level in two steps: the CaiD catalyzed hydration of crotonobetainyl-CoA to L-carnitinyl-CoA, followed by a CoA transfer from L-carnitinyl-CoA to crotonobetaine, catalyzed by CaiB. When gamma-butyrobetainyl-CoA was used as a cosubstrate (CoA donor), the first reaction is the CoA transfer. The optimal ratios of CaiB and CaiD during this hydration reaction, determined to be 4:1 when crotonobetainyl-CoA was used as cosubstrate and 5:1 when gamma-butyrobetainyl-CoA was used as cosubstrate, are different from that found for in vivo conditions (1:3).