Production of Coenzyme A by Sarcina lutea

To develop an efficient method for the production of coenzyme A (CoA), optimal conditions for its formation from pantothenic acid, cysteine, and adenine were studied. A number of microorganisms were screened for production of CoA. Strains belonging to the genera Sarcina, Bacillus, Microbacterium, Micrococcus, and Serratia accumulated CoA. Among these, Sarcina lutea was selected as the best organism, and the culture conditions for the production of CoA were investigated with this organism. Under optimal conditions, 600 mug of CoA per ml was accumulated in the culture broth. CoA was readily isolated in high purity by the use of charcoal, diethylaminoethyl-cellulose, Sephadex G-25, and Dowex-50. Yields of isolated CoA were over 33% from culture broth.     

Synthesis of S-Behenyl Coenzyme A

The synthesis of behenyl CoA by the reaction of coenzyme A with N-hydroxysuecinimide ester of behenic acid is reported. This method gives better yields of behenyl CoA when compared to the acid chloride or mixed anhydride method where side reactions seem to cause a decrease in yield.     

Microbial Synthesis of Coenzyme A Intermediates

Greater production of pantothenic acid 4′-phosphate and pantetheine 4′-phosphate by a microorganism were described. The incubation of pantothenic acid and adenosine 5′-triphosphate with resting cells of Brevibacterium ammoniagenes IFO 12071 gave pantothenic acid-4′-phosphate in a high yield. Cultivation of the organism with pantothenic acid and 5′adenylic acid also gave pantothenic acid 4′-phosphate in a high yield. In a similar fashion pantetheine 4′-phosphate was readily obtained in a good yield. The products were identified chemically and enzymatically.     

Coenzyme A: back in action

Coenzyme A (CoA) is a ubiquitous essential cofactor that plays a central role in the metabolism of carboxylic acids, including short- and long-chain fatty acids. In the last few years, all of the genes encoding the CoA biosynthetic enzymes have been identified and the structures of several proteins in the pathway have been determined. CoA is assembled in five steps from pantothenic acid and pathway intermediates are common to both prokaryotes and eukaryotes. In spite of the identical biochemistry, remarkable sequence differences among some of the prokaryotic and eukaryotic enzymes have been revealed by comparative genomics. Renewed interest in CoA has arisen from the realization that the biosynthetic pathway is a target for antibacterial drug discovery and from the unexpected association of a human neurodegenerative disorder with mutations in pantothenate kinase. The purpose of this review is to integrate previous knowledge with the most recent findings in the genetics, enzymology and regulation of CoA biosynthesis in bacteria, plants and mammals.     

A radioisotopic method for the determination of acetoacetyl Coenzyme A with 3-hydroxy-3-methylglutaryl Coenzyme A synthase

A simple and sensitive assay for the quantitative determination of acetoacetyl-CoA (AcAc-CoA) in liver and heart is described. The method is based on incorporation of [¹⁴C]acetyl-CoA into acid-stable nonvolatile material in the presence of avian HMG-CoA synthase. The specificity of this procedure for the measurement of AcAc-CoA was demonstrated by pretreating tissue extracts with 3-hydroxyacyl-CoA dehydrogenase or CoA transferase from Escherichia coli to deplete. AcAc-CoA prior to assay. Acid-stable nonvolatile ¹⁴C activity measured in the assay was proportional to the amount of tissue extract added. Satisfactory recovery of AcAc-CoA added at the initial extraction step further validated this procedure. This radioactive assay for acetoacetyl-CoA using a highly purified avian 3-hydroxy-3-methylglutaryl-CoA synthase has the advantages of both extreme specificity for AcAc-CoA as substrate and high sensitivity, facilitating the determination of this metabolite under a variety of physiological conditions.     

Acyl Coenzyme A Synthetase Regulation: Putative Role in Long‐Chain Acyl Coenzyme A Partitioning

Objective: Long‐chain acyl coenzyme A synthetase (ACSL) converts free fatty acids (FFAs) into their metabolizable long‐chain acyl coenzyme A (LC‐CoA) derivatives that are essential for FFA conversion to CO₂, triglycerides, or complex lipids. ACSL‐1 is highly expressed in adipose tissue with broad substrate specificity. We tested the hypothesis that ACSL localization, and resulting local generation of LC‐CoA, regulates FFA partitioning. Research Methods and Procedures: These studies used cell fractionation of rat adipocytes to measure ACSL activity and mass and compared cells from young, mature, fed, fasted, and diabetic rats. Functional studies included measurement of FFA oxidation, complex lipid synthesis, and LC‐CoA levels. Results: High ACSL specific activity was expressed in the mitochondria/nuclei (M/N), high‐density microsomes (HDM), low‐density microsomes (LDM), and plasma membrane (PM) fractions. We show here that, during fasting, total FFA oxidation increased, and, although total ACSL activity decreased, a greater percentage of activity (43 ± 1.5%) was associated with the M/N fraction than in the fed state (23 ± 0.3%). In the fed state, more ACSL activity (34 ± 0.5%) was associated with the HDM than in the fasted state (25 ± 0.9%), concurrent with increased triglyceride formation from FFA. Insulin increased LC‐CoA and ACSL activity associated with the PM. The changes in ACSL activity in response to insulin were associated with only minor changes in mass as determined by Western blotting. Discussion: It is hypothesized that ACSL plays an important role in targeting FFA to specific metabolic pathways or acylation sites in the cell, thus acting as an important control mechanism in fuel partitioning. Localization of ACSL at the PM may serve to decrease FFA efflux and trap FFA within the cell as LC‐CoA.     

Hydroxymethylglutaryl Coenzyme A Reductase in Fusarium oxysporum

Both growth and theanine accumulation in tea callus cultures were improved by the combination of 4 mg/liter benzyladenine and 2mg/liter indol-3-butyric acid, but were strongly inhibited by the addition of 2,4-dichlorophenoxyacetic acid. The optimum initial concentration of carbon source was 30g/liter sucrose. Upon the addition of more than 30 g/liter of sucrose, the callus fresh weight was increased, but the theanine formation was not improved.     

A New Preparation Method of Coenzyme A with Microorganisms

The structure of aggregates formed by heating dilute BSA solution was analyzed with the fractal concept using light scattering methods. BSA was dissolved in HEPES buffer of pH 7.0 and acetate buffer of pH 5.1 to 0.1% and 0.001% solutions, respectively, and heated at 95°C, varying the heating time t_a. The fractal dimension D_f of the aggregate in the solution was evaluated from static light scattering experiments. The polydispersity exponent τ and the average hydrodynamic radius <R_h> of the aggregates were calculated from dynamic light scattering experiments using master curves obtained by Klein et al. The values of D_f and τ of heat-induced aggregates of BSA at pH 7.0 were about 2.1 and 1.5, respectively, the values of which agreed with those predicted by the reaction-limited cluster–cluster aggregation (RLCCA) model. On the other hand, D_f of heat-induced aggregates at pH 5.1 was about 1.8, which agreed with that predicted by the diffusion-limited cluster–cluster aggregation (DLCCA) model. The dependence of <R_h> for the sample of pH 7.0 on t_a was similar to that of the polystyrene colloids reported previously.     

A New Process for the Production of Coenzyme A

A new process has been described for the preparation of coenzyme A of high purity from the cultured broth of Brevibacterium ammoniagenes IFO 12071. The product was obtained in a high yield by the use of Duolite S–30, charcoal, and Dowex 1×2, and identified chemically and enzymatically. This method is simple, rapid, and compact, requires no special equipment, and has been shown to be adaptable for preparing large amounts of highly pure coenzyme A.     

Production of Coenzyme A by a Mutant of Brevibacterium ammoniagenes Resistant to Oxypantetheine

For improved production of coenzyme A (CoA), a mutant of Brevibacterium ammoniagenes IFO127071 resistant to oxypantetheine, the corresponding oxygen analog of pantetheine, was obtained. In the mutant, activity of pantothenate kinase (EC 2.7.1.33), the first-step enzyme for the biosynthesis of CoA from pantothenic acid, l-cysteine, and ATP, was about threefold higher than that in the parent strain. As the main regulation mechanism of CoA biosynthesis in this bacterium is negative feedback inhibition of pantothenate kinase by CoA, the mutant is very useful as a catalyst for practical production of CoA. When added to culture broth of the mutant, pantothenate, l-cysteine, and AMP gave 9.3 mg of CoA per ml. With pantetheine and AMP, 11.5 mg of CoA per ml accumulated. These values were about threefold higher than those with the parent strain, and more than 70% of the added AMP was converted to CoA.     

Coenzyme A and the Malaria Parasite Plasmodium lophurae

The extracellular development of Plasmodium lophurae in vitro was favored by the addition, to the erythrocyte-extract medium, of a coenzyme A (CoA) preparation of about 75% purity. The effect of CoA was the same regardless of the concentration of free pantothenate in the medium, indicating that the parasites require the complete coenzyme rather than its pantothenic acid moiety. Erythrocyte extracts were found to contain enzymes which hydrolyzed added CoA at a rate such that 8 units per ml. of coenzyme was only slightly destroyed after 3 hours'incubation but almost completely destroyed after 18 hours'incubation at 40°C. The CoA content of erythrocytes from chickens or ducks heavily infected with P. lophurae was about twice as high as that of erythrocytes from uninfected birds. The increased CoA was associated with the parasites, an observation suggesting that malaria parasites can accumulate this essential growth factor which they cannot synthesize. The CoA concentration in the livers of infected chickens was approximately 40% lower than that in the livers of control chickens. The livers of ducks on the 6th day of infection had a slightly lower CoA concentration than those of uninfected ducks. This depletion in CoA, together with the depletion in biotin previously demonstrated in P. lophurae infection, may play a role in the pathology of this infection.     

Coenzyme A biosynthesis: an antimicrobial drug target

Pantothenic acid, a precursor of coenzyme A (CoA), is essential for the growth of pathogenic microorganisms. Since the structure of pantothenic acid was determined, many analogues of this essential metabolite have been prepared. Several have been demonstrated to exert an antimicrobial effect against a range of microorganisms by inhibiting the utilization of pantothenic acid, validating pantothenic acid utilization as a potential novel antimicrobial drug target. This review commences with an overview of the mechanisms by which various microorganisms acquire the pantothenic acid they require for growth, and the universal CoA biosynthesis pathway by which pantothenic acid is converted into CoA. A detailed survey of studies that have investigated the inhibitory activity of analogues of pantothenic acid and other precursors of CoA follows. The potential of inhibitors of both pantothenic acid utilization and biosynthesis as novel antibacterial, antifungal and antimalarial agents is discussed, focusing on inhibitors and substrates of pantothenate kinase, the enzyme catalysing the rate-limiting step of CoA biosynthesis in many organisms. The best strategies are considered for identifying inhibitors of pantothenic acid utilization and biosynthesis that are potent and selective inhibitors of microbial growth and that may be suitable for use as chemotherapeutic agents in humans.     

Coenzyme A fuels T cell anti-tumor immunity

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