Substrate specificity of human carnitine acetyltransferase: Implications for fatty acid and branched-chain amino acid metabolism

Carnitine acyltransferases catalyze the reversible conversion of acyl-CoAs into acylcarnitine esters. This family includes the mitochondrial enzymes carnitine palmitoyltransferase 2 (CPT2) and carnitine acetyltransferase (CrAT). CPT2 is part of the carnitine shuttle that is necessary to import fatty acids into mitochondria and catalyzes the conversion of acylcarnitines into acyl-CoAs. In addition, when mitochondrial fatty acid β-oxidation is impaired, CPT2 is able to catalyze the reverse reaction and converts accumulating long- and medium-chain acyl-CoAs into acylcarnitines for export from the matrix to the cytosol. However, CPT2 is inactive with short-chain acyl-CoAs and intermediates of the branched-chain amino acid oxidation pathway (BCAAO). In order to explore the origin of short-chain and branched-chain acylcarnitines that may accumulate in various organic acidemias, we performed substrate specificity studies using purified recombinant human CrAT. Various saturated, unsaturated and branched-chain acyl-CoA esters were tested and the synthesized acylcarnitines were quantified by ESI-MS/MS. We show that CrAT converts short- and medium-chain acyl-CoAs (C2 to C10-CoA), whereas no activity was observed with long-chain species. Trans-2-enoyl-CoA intermediates were found to be poor substrates for this enzyme. Furthermore, CrAT turned out to be active towards some but not all the BCAAO intermediates tested and no activity was found with dicarboxylic acyl-CoA esters. This suggests the existence of another enzyme able to handle the acyl-CoAs that are not substrates for CrAT and CPT2, but for which the corresponding acylcarnitines are well recognized as diagnostic markers in inborn errors of metabolism.     

Quantitation of methylmalonic acid and other dicarboxylic acids in normal serum and urine using capillary gas chromatography-mass spectrometry

Methylmalonic acid, succinic acid, and other dicarboxylic acids have been extracted and partially purified from serum and urine using ether extraction and high-performance liquid chromatography. The t-butyldimethylsilyl derivatives were prepared and analyzed using capillary gas chromatography-mass spectrometry with selected ion monitoring. The addition of [methyl-2H3]methylmalonic acid and [1,4-13C2]succinic acid to the starting samples made it possible to quantitate these two dicarboxylic acids. Normal ranges for methylmalonic acid and succinic acid were determined in human and rat serum and in human urine. The utilization of other internal standards would make it possible to quantitate malonic, dimethylmalonic, ethylmalonic, methylsuccinic, glutaric, and other dicarboxylic acids.     

Metabolic origin of urinary 3-hydroxy dicarboxylic acids

3-Hydroxy dicarboxylic acids with chain lengths ranging from 6 to 14 carbons are excreted in human urine. The urinary excretion of these acids is increased in conditions of increased mobilization of fatty acids or inhibited fatty acid oxidation. Similar urinary profiles of 3-hydroxy dicarboxylic acids were also observed in fasting rats. The metabolic genesis of these urinary 3-hydroxy dicarboxylic acids was investigated in vitro with rat liver postmitochondrial and mitochondrial fractions. 3-Hydroxy monocarboxylic acids ranging from 3-hydroxyhexanoic acid to 3-hydroxyhexadecanoic acid were synthesized. In the rat liver postmitochondrial fraction fortified with NADPH, these 3-hydroxy fatty acids with carbon chains equal to or longer than 10 were oxidized to (omega - 1)- and omega-hydroxy metabolites as well as to the corresponding 3-hydroxy dicarboxylic acids. 3-Hydroxyhexanoic (3OHMC6) and 3-hydroxyoctanoic (3OHMC8) acids were not metabolized. Upon the addition of mitochondria together with ATP, CoA, carnitine, and MgCl2, the 3-hydroxy dicarboxylic acids were converted to 3-hydroxyoctanedioic, trans-2-hexenedioic, suberic, and adipic acids. In the urine of children with elevated 3-hydroxy dicarboxylic acid levels, 3OHMC6, 3OHMC8, 3-hydroxydecanoic, 3,10-dihydroxydecanoic, 3,9-dihydroxydecanoic, and 3,11-dihydroxydodecanoic acids were identified. On the basis of these data, we propose that the urinary 3-hydroxy dicarboxylic acids are derived from the omega-oxidation of 3-hydroxy fatty acids and the subsequent beta-oxidation of longer chain 3-hydroxy dicarboxylic acids. These urinary 3-hydroxy dicarboxylic acids are not derived from the beta-oxidation of unsubstituted dicarboxylic acids.     

Separation of acylcarnitines from biological samples using high-performance liquid chromatography

A reverse-phase high-performance liquid chromatography technique to separate carnitine and acylcarnitines from a biological matrix is described. The method utilizes a step gradient to provide baseline resolution of acylcarnitines (individually or by class) for subsequent quantification using a sensitive radioenzymatic assay. The method requires minimal sample preparation and prevents any contamination among groups of acylcarnitines. This technique has been applied to liver tissues of rats obtained under a variety of conditions. These studies demonstrate the validity and utility of the HPLC method while confirming the applicability of the perchloric acid fractionation of acylcarnitines by functional class. The present HPLC method permits resolution of long-chain acylcarnitines in the presence of large excess concentrations of carnitine and short-chain acylcarnitines (coelution of unesterified carnitine with long-chain acylcarnitines less than or equal to 0.05%). Thus, the method will be of use in the study of acylcarnitines in biological systems over a broad spectrum of metabolic conditions.     

Gas chromatography-mass spectrometry profile of urinary organic acids of Wistar rats orally treated with ozonized unsaturated triglycerides and ozonized sunflower oil

The main products in the ozonolysis of unsaturated triglycerides or vegetable oils are peroxides, aldehydes, Criegee ozonides and carboxylic acids. Some of these compounds are present in different concentrations in the biological fluids. The aim of this work is to study, using gas chromatography-mass spectrometry (GC-MS), the organic acid excretion in urine of rats orally treated with ozonized sunflower oil (OSO), ozonized triolein or ozonized trilinolein. Oral administration of OSO to Wistar rats has produced changes in the urinary content of dicarboxylic organic acids. Among others heptanedioic (pimelic acid) and nonanedioic acids (azelaic acid) were the major increased dicarboxylic acids found. The urinary dicarboxylic acid profiles of rats which received ozonized triolein only showed an increase in heptanedioic and nonanedioic acids. However, when ozonized trilinolein is applied, the profile is similar to that obtained when OSO is administered. A biochemical mechanism is proposed to explain the formation of dicarboxylic acids from ozonated unsaturated triglycerides.     

Dicarboxylic acid transport in membrane vesicles from Bacillus subtilis.

Membrane vesicles isolated from Bacillus subtilis W23 catalyze active transport of the C4 dicarboxylic acids L-malate, fumarate, and succinate under aerobic conditions in the presence of the electron donor reduced beta-nicotinamide adenine dinucleotide or the non-physiological electron donor system ascorbate-phenazine methosulfate. The dicarboxylic acids are accumulated in unmodified form. Inhibitors of the respiratory chain, sulfhydryl reagents, and uncoupling agents inhibit the accumulation of the dicarboxylic acids. The affinity constants for transport of L-malate, fumarate, and succinate are 13.5, 7.5, and 4.3 muM, respectively; these values are severalfold lower than those reported previously for whole cells. Active transport of these dicarboxylic acids occurs via one highly specific transport system as is indicated by the following observations. (i) Each dicarboxylic acid inhibits the transport of the other two dicarboxylic acids competitively. (ii) The affinity constants determined for the inhibitory action are very similar to those determined for the transport process. (iii) Each dicarboxylic acid exchanges rapidly with a previously accumulated dicarboxylic acid. (iv) Other metabolically and structurally related compounds do not inhibit transport of these dicarboxylic acids significantly, except for L-aspartate and L-glutamate. However, transport of these dicarboxylic amino acids is mediated by independent system because membrane vesicles from B. subtilis 60346, lacking functional dicarboxylic amino acid transport activity, accumulate the C4 dicarboxylic acids at even higher rates than vesicles from B. subtilis W 23. (v) A constant ratio exists between the initial rates of transport of L-malate, fumarate, and succinate in all membrane vesicle preparations isolated from cells grown on various media. This high-affinity dicarboxylic acid transport system seems to be present constitutively in B. subtilis W23.     

Isolation and identification of alpha-methyloctanylcarnitines from human urine

Medium-chain acylcarnitines were isolated from human urine using a combination of chloroform-methanol extraction, silicic acid column and molecular sieving chromatography and preparative HPLC. Three purified acylcarnitines were analyzed by fast atom bombardment mass spectrometry and were also saponified and the free fatty acids analyzed by gas chromatography and mass spectrometry. Combined electron impact mass spectrometry and fast atom bombardment mass spectrometry and periodate oxidation for location of double bonds, demonstrated the occurrence of delta 6-octenylcarnitine, 2-methyloctanylcarnitine and 2-methyl-delta 6-octenylcarnitine. These acylcarnitines were present in the thirteen urines obtained from normal humans, but were not detected in urines from three individuals who had been on total parenteral nutrition for more than a year. The occurrence of alpha-methyl medium-chain acylcarnitines in human urine indicates a role for carnitine in excretion (detoxification) of these acyl derivatives.     

Relationship between acid-soluble carnitine and coenzyme A pools in vivo

The relationship between the acid-soluble carnitine and coenzyme A pools was studied in fed and 24-h-starved rats after carnitine administration. Carnitine given by intravenous injection at a dose of 60μmol/100g body wt. was integrated into the animal's endogenous carnitine pool. Large amounts of acylcarnitines appeared in the plasma and liver within 5min of carnitine injection. Differences in acid-soluble acylcarnitine concentrations were observed between fed and starved rats after injection and reflected the acylcarnitine/carnitine relationship seen in the endogenous carnitine pool of the two metabolic states. Thus, a larger acylcarnitine production was seen in starved animals and indicated a greater source of accessible acyl-CoA molecules. In addition to changes in the amount of acylcarnitines present, the specific acyl groups present also varied between groups of animals. Acetylcarnitine made up 37 and 53% of liver acid-soluble acylcarnitines in uninjected fed and starved animals respectively. At 5min after carnitine injection hepatic acid-soluble acylcarnitines were 41 and 73% in the form of acetylcarnitine in fed and starved rats respectively. Despite these large changes in carnitine and acylcarnitines, no changes were observed in plasma non-esterified fatty acid or β-hydroxybutyrate concentrations in either fed or starved rats. Additionally, measurement of acetyl-CoA, coenzyme A, total acid-soluble CoA and acid-insoluble CoA demonstrated that the hepatic CoA pool was resistant to carnitine-induced changes. This lack of change in the hepatic CoA pool or ketone-body production while acyl groups are shunted from acyl-CoA molecules to acylcarnitines suggests a low flux through the carnitine pool compared with the CoA pool. These results support the concept that the carnitine/acid-soluble acylcarnitine pool reflects changes in, rather than inducing changes in, the hepatic CoA/acyl-CoA pool.     

Isolation and Identification of α-Methyloctanylcarnitines from Human Urine

Medium-chain acylcarnitines were isolated from human urine using a combination of chloroform-methanol extraction, silicic acid column and molecular sieving chromatography and preparative HPLC. Three purified acylcarnitines were analyzed by fast atom bombardment mass spectrometry and were also saponified and the free fatty acids analyzed by gas chromatography and mass spectrometry. Combined electron inpact mass spectrometry and fast atom bombardment mass spectrometry and periodate oxidation for location of double bonds, demonstrated the occurrence of δ⁶-octenylcarnitine, 2-methyloct-anylcarnitine and 2-methyl-δ⁶-octenylcarnitine. These acylcarnitines were present in the thirteen urines obtained from normal humans, but were not detected in urines from three individuals who had been on total parenteral nutrition for more than a year. The occurrence of α-methyl medium-chain acylcarnitines in human urine indicates a role for carnitine in excretion (detoxification) of these acyl derivatives.     

Properties of an inducible C 4 -dicarboxylic acid transport system in Bacillus subtilis

The transport of the tricarboxylic acid cycle C(4)-dicarboxylic acids was studied in both the wild-type strain and tricarboxylic acid cycle mutants of Bacillus subtilis. Active transport of malate, fumarate, and succinate was found to be inducible by these dicarboxylic acids or by precursors to them, whereas glucose or closely related metabolites catabolite-repressed their uptake. l-Malate was found to be the best dicarboxylic acid transport inducer in succinic dehydrogenase, fumarase, and malic dehydrogenase mutants. Succinate and fumarate are accumulated over 100-fold in succinic dehydrogenase and fumarase mutants, respectively, whereas mutants lacking malate dehydrogenase were unable to accumulate significant quantities of the C(4)-dicarboxylic acids. The stereospecificity of this transport system was studied from a comparison of the rates of competitive inhibition of both succinate uptake and efflux in a succinate dehydrogenase mutant by utilizing thirty dicarboxylic acid analogues. The system was specific for the C(4)-dicarboxylic acids of the tricarboxylic acid cycle, neither citrate nor alpha-ketoglutarate were effective competitive inhibitors. Of a wide variety of metabolic inhibitors tested, inhibiors of oxidative phosphorylation and of the formation of proton gradients were the most potent inhibitors of transport. From the kinetics of dicarboxylic acid transport (K(m) approximately 10(-4) M for succinate or fumarate in succinic acid dehydrogenase and fumarase mutants) and from the competitive inhibition studies, it was concluded that an inducible dicarboxylic acid transport system mediates the entry of malate, fumarate, or succinate into B. subtilis. Mutants devoid of alpha-ketoglutarate dehydrogenase were shown to accumulate both alpha-ketoglutarate and glutamate, and these metabolites subsequently inhibited the transport of all the C(4)-dicarboxylic acids, suggesting a regulatory role.     

Formation and degradation of dicarboxylic acids in relation to alterations in fatty acid oxidation in rats.

Dicarboxylic acids are excreted in urine when fatty acid oxidation is increased (ketosis) or inhibited (defects in beta-oxidation) and in Reye's syndrome. omega-Hydroxylation and omega-oxidation of C6-C12 fatty acids were measured by mass spectrometry in rat liver microsomes and homogenates, and beta-oxidation of the dicarboxylic acids in liver homogenates and isolated mitochondria and peroxisomes. Medium-chain fatty acids formed large amounts of medium-chain dicarboxylic acids, which were easily beta-oxidized both in vitro and in vivo, in contrast to the long-chain C16-dicarboxylic acid, which was toxic to starved rats. Increment of fatty acid oxidation in rats by starvation or diabetes increased C6:C10 dicarboxylic acid ratio in rats fed medium-chain triacylglycerols, and increased short-chain dicarboxylic acid excretion in urine in rats fed medium-chain dicarboxylic acids. Valproate, which inhibits fatty acid oxidation and may induce Reye like syndromes, caused the pattern of C6-C10-dicarboxylic aciduria seen in beta-oxidation defects, but only in starved rats. It is suggested, that the origin of urinary short-chain dicarboxylic acids is omega-oxidized medium-chain fatty acids, which after peroxisomal beta-oxidation accumulate as C6-C8-dicarboxylic acids. C10-C12-dicarboxylic acids were also metabolized in the mitochondria, but did not accumulate as C6-C8-dicarboxylic acids, indicating that beta-oxidation was completed beyond the level of adipyl CoA.     

Long-chain Acylcarnitines Reduce Lung Function by Inhibiting Pulmonary Surfactant

The role of mitochondrial energy metabolism in maintaining lung function is not understood. We previously observed reduced lung function in mice lacking the fatty acid oxidation enzyme long-chain acyl-CoA dehydrogenase (LCAD). Here, we demonstrate that long-chain acylcarnitines, a class of lipids secreted by mitochondria when metabolism is inhibited, accumulate at the air-fluid interface in LCAD^−/− lungs. Acylcarnitine accumulation is exacerbated by stress such as influenza infection or by dietary supplementation with l-carnitine. Long-chain acylcarnitines co-localize with pulmonary surfactant, a unique film of phospholipids and proteins that reduces surface tension and prevents alveolar collapse during breathing. In vitro, the long-chain species palmitoylcarnitine directly inhibits the surface adsorption of pulmonary surfactant as well as its ability to reduce surface tension. Treatment of LCAD^−/− mice with mildronate, a drug that inhibits carnitine synthesis, eliminates acylcarnitines and improves lung function. Finally, acylcarnitines are detectable in normal human lavage fluid. Thus, long-chain acylcarnitines may represent a risk factor for lung injury in humans with dysfunctional fatty acid oxidation.     

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency: diagnosis by acylcarnitine analysis in blood.

Medium-chain acyl-coenzyme A dehydrogenase (MCAD) deficiency is a disorder of fatty acid catabolism, with autosomal recessive inheritance. The disease is characterized by episodic illness associated with potentially fatal hypoglycemia and has a relatively high frequency. A rapid and reliable method for the diagnosis of MCAD deficiency is highly desirable. Analysis of specific acylcarnitines was performed by isotope-dilution tandem mass spectrometry on plasma or whole blood samples from 62 patients with MCAD deficiency. Acylcarnitines were also analyzed in 42 unaffected relatives of patients with MCAD deficiency and in other groups of patients having elevated plasma C8 acylcarnitine, consisting of 32 receiving valproic acid, 9 receiving medium-chain triglyceride supplement, 4 having multiple acyl-coenzyme A dehydrogenase deficiency, and 8 others with various etiologies. Criteria for the unequivocal diagnosis of MCAD deficiency by acylcarnitine analysis are an elevated C8-acylcarnitine concentration (> 0.3 microM), a ratio of C8/C10 acylcarnitines of > 5, and lack of elevated species of chain length > C10. These criteria were not influenced by clinical state, carnitine treatment, or underlying genetic mutation, and no false-positive or false-negative results were obtained. The same criteria were also successfully applied to profiles from neonatal blood spots retrieved from the original Guthrie cards of eight patients. Diagnosis of MCAD deficiency can therefore be made reliably through the analysis of acylcarnitines in blood, including presymptomatic neonatal recognition. Tandem mass spectrometry is a convenient method for fast and accurate determination of all relevant acylcarnitine species.     

Hydrocarbon Metabolism by Brevibacterium erythrogenes: Normal and Branched Alkanes

Branched- and straight-chain alkanes are metabolized by Brevibacterium erythrogenes by means of two distinct pathways. Normal alkanes (e.g., n-pentadecane) are degraded, after terminal oxidation, by the beta-oxidation system operational in fatty acid catabolism. Branched alkanes like pristane (2,6,10,14-tetramethylpentadecane) and 2-methylundecane are degraded as dicarboxylic acids, which also undergo beta-oxidation. Pristane-derived intermediates are observed to accumulate, with time, as a series of dicarboxylic acids. This dicarboxylic acid pathway is not observed in the presence of normal alkanes. Release of (14)CO(2) from [1-(14)C]pristane is delayed, or entirely inhibited, in the presence of n-hexadecane, whereas CO(2) release from n-hexadecane remains unaffected. These results suggest an inducible dicarboxylic acid pathway for degradation of branched-chain alkanes.     

Carnitine is associated with fatty acid metabolism in plants

The finding of acylcarnitines alongside free carnitine in Arabidopsis thaliana and other plant species, using tandem mass spectrometry coupled to liquid chromatography shows a link between carnitine and plant fatty acid metabolism. Moreover the occurrence of both medium- and long-chain acylcarnitines suggests that carnitine is connected to diverse fatty acid metabolic pathways in plant tissues. The carnitine and acylcarnitine contents in plant tissues are respectively a hundred and a thousand times lower than in animal tissues, and acylcarnitines represent less than 2% of the total carnitine pool whereas this percentage reaches 30% in animal tissues. These results suggest that carnitine plays a lesser role in lipid metabolism in plants than it does in animals.     

Factors contributing to the inhibition of aspartate aminotransferase by dicarboxylic acids.

At pH 8.0 aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) reacts with the modified substrate, erythro-beta-hydroxy-L-aspartate, to form a mixture of enzyme-substrate complexes absorbing at 492 nm. A variety of dicarboxylic acids were studied spectrophotometrically as competitive inhibitors of this reaction. All of the inhibitory dicarboxylic acids form a complex with the enzyme, absorbing at 362 nm. In addition, some of the dicarboxylic acids form a protonated complex absorbing at about 435 nm. This complex, which is the conjugate acid of that absorbing at 362 nm, is formed only by those dicarboxylic acids which can assume a configuration in which the two carboxyl groups are positioned as in maleic acid. Bulky substituents, such as aromatic rings or even methyl groups, prevent the formation of the protonated complex, presumably because of steric restrictions at the active site. Substitution of the central carbon atom of glutaric acid by heteroatoms of increasing charge density results in a progressive decrease in inhibitory effectiveness, at pH 8, primarily due to a loss of this pH-dependent stabilization of the enzyme-dicarboxylic acid complex. Acids with an aromatic ring are among the most potent dicarboxylic acid inhibitors of this enzyme in spite of the fact that they do not undergo the pH-dependent stabilization of their enzyme complexes. From these observations it was concluded that the affinity of aspartate aminotransferase for dicarboxylic acids is determined as much by the mechanism of binding as by the solvation and steric effects.     

Dicarboxylic acids with limited numbers of hydrocarbons stabilize cell membrane and increase osmotic resistance in rat erythrocytes

We examined the effect of dicarboxylic acids having 0 to 6 hydrocarbons and their corresponding monocarboxylic or tricarboxylic acids in changing the osmotic fragility (OF) in rat red blood cells (RBCs). Malonic, succinic, glutaric and adipic acids, which are dicarboxylic acids with 1, 2, 3 and 4 straight hydrocarbons located between two carboxylic groups, decreased the OF in a concentration-dependent manner. Other long-chain dicarboxylic acids did not change the OF in rat RBCs. The benzoic acid derivatives, isophthalic and terephthalic acids, but not phthalic acid, decreased the OF in a concentration-dependent manner. Benzene-1,2,3-tricarboxylic acid, but not benzene-1,3,5-tricarboxylic acid, also decreased the OF in rat RBCs. On the other hand, monocarboxylic acids possessing 2 to 7 straight hydrocarbons and benzoic acid increased the OF in rat RBCs. In short-chain dicarboxylic acids, a limited number of hydrocarbons between the two carboxylic groups are thought to form a V- or U-shaped structure and interact with phospholipids in the RBC membrane. In benzene dicarboxylic and tricarboxylic acids, a part of benzene nucleus between the two carboxylic groups is thought to enter the plasma membrane and act on acyl-chain in phospholipids in the RBC membrane. For dicarboxylic and tricarboxylic acids, limited numbers of hydrocarbons in molecules are speculated to enter the RBC membrane with the hydrophilic carboxylic groups remaining outside, stabilizing the structure of the cell membrane and resulting in an increase in osmotic resistance in rat RBCs.     

Abnormal urinary excretion of unsaturated dicarboxylic acids in patients with medium-chain acyl-CoA dehydrogenase deficiency

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most frequently described metabolic disorder of fatty acid oxidation in humans. Acute episodes are usually characterized biochemically by the appearance of nonketotic dicarboxylic aciduria. In addition, other abnormal metabolites, such as suberylglycine, n-hexanoylglycine, 3-phenylpropionylglycine, and octanoylcarnitine, are excreted in the urine. Urinary organic acids were determined using dual capillary column gas-liquid chromatography and gas-liquid chromatography/mass spectrometry. In three cases of MCAD deficiency we observed a disproportionate increase in the excretion of unsaturated dicarboxylic acids compared to either fasting control children with expected ketotic dicarboxylic aciduria or patients with nonketotic dicarboxylic aciduria not associated with MCAD deficiency. The most significant increase was in the urinary excretion of cis-4-decendioic acid. Additionally, the urinary excretions of cis-3-octenedioic and cis-5-decenedioic acids were slightly decreased whereas the excretion of cis-5-dodecenedioic acid was increased. These data are consistent with the notion that as a result of MCAD deficiency the metabolic oxidation of unsaturated fatty acids such as linoleate and oleate is inhibited more than saturated fatty acids.     

N-Heterocyclic dicarboxylic acids: Broad-spectrum inhibitors of metallo-β-lactamases with co-antibacterial effect against antibiotic-resistant bacteria

In an effort to identify novel, broad-spectrum inhibitors against the metallo-β-lactamases (MβLs), several N-heterocyclic derivatives were tested as inhibitors of MβLs CcrA, ImiS, and L1, which are representative enzymes from the distinct MβL subclasses. Three N-heterocyclic dicarboxylic acid derivatives were competitive inhibitors of CcrA and L1, exhibiting K(i) values ?2μM, while only 2,4-thiazolidinedicarboxylic acid (1b) was a competitive inhibitor of ImiS. Two 2-mercapto-1,3,4-thiadiazole derivatives were noncompetitive inhibitors of CcrA and ImiS, exhibiting K(i) values <7μM; however, these same compounds did not inhibit L1. Two 2-mercapto-1,3,4-triazole derivatives were shown not to inhibit any of the tested MβLs. The N-heterocyclic derivatives were tested for antibacterial activity by examining the MIC values for existing antibiotics in the presence/absence of these derivatives. Consistent with the steady-state inhibition data, the inclusion of three N-heterocyclic dicarboxylic acid derivatives resulted in lower MIC values when using Escherichia coli BL21(DE3) cells containing the CcrA or L1 plasmids or Klebsiella pneumoniae (ATCC 700603), while 1b was the only dicarboxylic acid derivative to lower the MIC value of E. coli cells containing the ImiS plasmid. Inclusion of the 2-mercapto-1,3,4-thiadiazole derivatives resulted in lower MIC values for E. coli cells containing ImiS or L1 plasmids; however, these derivatives did not alter the MIC values for K. pneumoniae or E. coli cells containing the L1 plasmid. None of the N-heterocyclic derivatives affected the MIC of two methicillin resistant Staphylococcus aureus (MRSA) strains. Taken together, these studies demonstrate that N-heterocyclic dicarboxylic acids 1a-c and pyridylmercaptothiadiazoles 2a,b are good scaffolds for future broad-spectrum inhibitors of the MβLs.     

Production of dicarboxylic acids from novel unsaturated fatty acids by laccase-catalyzed oxidative cleavage

The establishment of renewable biofuel and chemical production is desirable because of global warming and the exhaustion of petroleum reserves. Sebacic acid (decanedioic acid), the material of 6,10-nylon, is produced from ricinoleic acid, a carbon-neutral material, but the process is not eco-friendly because of its energy requirements. Laccase-catalyzing oxidative cleavage of fatty acid was applied to the production of dicarboxylic acids using hydroxy and oxo fatty acids involved in the saturation metabolism of unsaturated fatty acids in Lactobacillus plantarum as substrates. Hydroxy or oxo fatty acids with a functional group near the carbon–carbon double bond were cleaved at the carbon–carbon double bond, hydroxy group, or carbonyl group by laccase and transformed into dicarboxylic acids. After 8 h, 0.58 mM of sebacic acid was produced from 1.6 mM of 10-oxo-cis-12,cis-15-octadecadienoic acid (αKetoA) with a conversion rate of 35% (mol/mol). This laccase-catalyzed enzymatic process is a promising method to produce dicarboxylic acids from biomass-derived fatty acids.