Studies on the Metabolism of Pantothenic Acid in Microorganisms

The ability of the formation of coenzyme A from pantothenic acid and cysteine in the presence of AMP or ATP was searched in yeasts and bacteria. The result of screening showed that the activity was found in several yeasts and the bacteria belonging to the genera Sarcina, Corynebacterium and Brevibacterium. Particularly, Brevibacterium ammoniagenes IFO 12071 (ATCC 6871) accumulated a large amount of coenzyme A.

Isolation of the reaction products, which were synthesized by Brevibacterium ammoniagenes IFO 12071, were carried out. The isolates were identified as coenzyme A, dephosphocoenzyme A and phosphopantothenic acid.

The possibility for the formation of coenzyme A in a larger amount from pantothenic acid and cysteine was investigated with baker’s yeast under the condition coupled with ATP-generating system.

Effect of various factors affecting the accumulation of coenzyme A was investigated. Among them, glucose concentration and inorganic phosphorus concentration were the most important factors for its accumulation. Coenzyme A was not accumulated without the phosphorylation of AMP to ATP. Several cationic surfactants stimulated the accumulation of coenzyme A.

The amount of coenzyme A accumulated reached about 200 μg per ml of the reaction mixture under the suitable reaction conditions employed.     

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.     

An Improved Method for the Fermentative Production of Coenzyme A from Pantothenic Acid,Cysteine, and 5′-AMP

The cultivation of Brevibacterium ammoniagenes IFO 12071 with pantothenic acid, cysteine, and 5′-adenylic acid gave coenzyme A in a high yield. The organism was stabilized by repeated single colony isolations. The culture conditions optimal for the production of coenzyme A were investigated, and the yield of coenzyme A in the culture broth reached more than 3 mg/ml.

The advantages and disadvantages of the present method were discussed by comparing them with our original dried cell method.     

Purification and Properties of Pantothenate Kinase from Brevibacterium ammoniagenes IFO 12071

Pantothenate kinase (ATP: pantothenate 4′-phosphotransferase, EC 2.7.1.33) was purified about 200-fold from the cell extract of Brevibacterium ammoniagenes IFO 12071 by ammonium sulfate fractionation, DEAE-cellulose chromatography, and Sephadex G-150 gel filtration. The purified enzyme gave a single band on polyacrylamide gel electrophoresis. The molecular weight was calculated approximately 45,000. The enzyme catalyzed the formation of pantothenic acid 4′-phosphate and ADP from pantothenate and ATP in the presence of Mg²⁺ ATP could be substituted for, partly, by ITP, GTP, and UTP. The enzyme phosphorylated not only pantothenate, but also pantothenoylcysteine, pantetheine, and pantothenyl alcohol. Apparent Km values were 6.7×10^?5 m for pantothenate, 3.5×10^?5 m for ATP, and 10^?3 m for Mg²⁺. The reaction was inhibited by the intermediates of CoA biosynthesis, of which CoA itself was a most effective inhibitor. Other properties of the enzyme were also investigated.     

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.     

The capacity of orotic acid production in pyrimidinedeficient mutants ofBrevibacterium ammoniagenes

Eight uracil-dependent mutants ofBrevibacterium ammoniagenes CCEB 364 and three mutants ofCorynebacterium sp. 9366 were checked for the production of precursors of nucleic acids. Four of the strains liberated into the medium a substantial amount of orotic acid. The production of orotic acid by a mutant ofBrevibacterium ammoniagenes (1043) was examined on mineral media containing varying amounts of glucose in the presence of uracil. The optimum concentration of glucose for the production of orotic acid was found to be 5–8%. On media to which natural substrates were added the orotic acid production increased substantially. The maximum production (6.5 g orotic acid/liter) was reached in a medium containing 0.5% yeast extract and 5% glucose; addition of uracil to this medium had no effect on the production. The maximum rate of production occurred between 24 and 72 h of fermentation. After this period the concentration of orotic acid in the medium decreases.     

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.     

Production of Nucleic Acid-Related Substances by Fermentative Processes

1. Suitable agar plate media were selected for isolation of nucleotide producing strains, by salvage synthesis, from natural sources. Since this agar medium contains a high concentration of phosphates, manganese and glucose, it is specific for these bacteria.

2. With this plate medium, 113 bacterial strains accumulating 5′inosinic acid (IMP) or IMP-like substances were isolated effectively from feces of a variety of birds and mammals and from soils.

Some of the strains isolated were recognized to accumulate other nucleotides, purine bases and sugars, such as guanine nucleotides, XMP, xanthine, ribulose or xylnlose, with or without hypoxanthine in the media.

3. Five strains of IMP accumulating bacteria were identified; two were classified as Brevibacteriurm, two as Corynebacterium and one as Arthrobacterium species by taxonomical studies. But their characteristics did not completely coincide with those of bacteria described in Bergey’s manual.

4. One of the IMP producing bacteria isolated, culture No. 21–26, actually consisted of two separate strains, namely No. 21–26–101 and No. 21–26–102. The highest production of IMP or guanine nucleotides was obtained, when each strain was inoculated together to the fermentation medium from each seed culture in the same inoculum size.

5. The nucleotide productions by No. 21–26–101 or No. 21–26–102 with authentic strains were examined by the mixed culture technique. It was found that production of IMP or guanine nucleotides by Brevibacterium ammoniagenes ATCC 6871 was stimulated remarkably in the presence of No. 21–26–102.     

Mitochondrial biosynthesis of coenzyme A.

All enzymes required for the biosynthesis of CoA from pantothenic acid are present in the particle-free supernatant fraction from rat liver. We now report that also mitochondria have the capacity for biosynthesis of CoA, with 4′-phosphopantetheine as the initial precursor. Rat liver mitochondria do not contain pantothenate kinase, 4′-phosphopantothenoyl-1-synthetase or 4′-phosphopantothenoyl-1-cysteine decarboxylase. Dephospho-CoA pyrophosphorylase and dephospho-CoA kinase are present in the inner mitochondrial membrane, however, at specific activities as high as in cytosol. K_m of mitochondrial dephospho-CoA kinase for dephospho-CoA is about 0.01 mmol/1, which is one order of magnitude lower than reported for the kinase from cytosol.     

Pentose Metabolism in Candida utilis

In attempts to obtain GMP producing strains, Brevibacterium ammoniagenes was treated with UV, N.T.G. or D.E.S. as a mutagen. Adenine-guanine requiring mutants were obtained from an adenine-requiring mutant of Brev. ammoniagenes, KY 3482–9 and two of them, presumably adenine-xanthine requiring mutants, were then reverted to mutants which required only adenine for their growth.

Although these revertants were not able to accumulate a copious amount of GMP, most of them and of adenine-guanine requiring mutants produced larger amounts of IMP than the parent adenine-requiring strain.

Effects of Mn²⁺ and purine bases in the medium on IMP production by these mutants were examined and IMP productivities of these mutants were compared with the parent strain under optimal conditions.

These mutagenic treatments were thus proved to be effective for the increase of de novo IMP production by Brev. ammoniagenes mutants.

Brevibacterium ammoniagenes ATCC 6872 accumulates 5′-GDP and -GTP, or 5′-ADP and -ATP together with GMP or AMP in nucleotide fermentation by salvage synthesis.

With cell free extract of this strain, transphosphorylating reactions of AMP or GMP were investigated.

ATP-AMP transphosphorylating enzyme(s) was partially purified to 21.7 fold with acid treatment, salting-out and column chromatography.

In ATP-AMP and ATP-GMP transphosphorylating reactins, optimal conditions were decided such as for concentrations of enzyme, of MgCl₂ and of phosphate donor, pH and cell age as the enzyme sources.

Specificities of phosphate donors and acceptors were examined with both the partially purified enzymes or the sonicate. AMP and GMP were phosphorylated by ATP rapidly, but IMP and XMP were not, therefore supporting our previous finding that Brev. ammoniagenes could not accumulated IDP, ITP, XDP and XTP in IMP and XMP fermentation, respectively.

Although ATP was the best donor for both AMP and GMP phosphorylations, other nucleoside triphosphates and PRPP were used as phosphate donors.

Furthermore, phosphorylation of ADP to ATP was investigated and possible mechanisms of nucleoside di- or triphosphates synthesis in the nucleotide fermentation were discussed.

From these results, it is suggested as a possible mechanism for nucleoside di- and triphosphate accumulation by Brev. Ammoniagenes, that a nucleoside monophosphate formed is phosphorylated to a nucleoside di-phosphate with ATP or other phosphate donors and then the nucleoside diphosphate is converted to a triphosphate with these phosphate donors.

Both AMP and GMP were transphosphorylated rapidly to the corresponding nucleoside-diphosphates and triphosphates by ATP and by other high energy phosphate compounds with cell free extracts of Brevibacterium ammoniagenes.

Some enzyme inhibitors, such as metals and PCMB were shown to inhibit the phosphorylations of AMP and GMP. Higher levels of ATP, ADP, GTP and GDP also inhibited the activity of the partially purified ATP-AMP transphosphorylating enzyme(s).

In guanine nucleotides fermentation by salvage synthesis with this strain, addition of these inhibitors to the medium increased the amounts of GMP and total guanine nucleotides accumulated.

On the contrary, supplement of xylene or of other organic solvents to the medium stimulated the accumulation of both GTP and total guanine compouuds in this fermentation. From enzymatic studies, these solvents are presumed to have the ability to change cell permeability.

Such findings give an effective method for controlling the amounts of nucleotides accumulated in these fermentations.     

Secretion of Streptomyces mobaraensis pro-transglutaminase by coryneform bacteria

We previously reported on the secretion of Streptomyces mobaraensis transglutaminase by Corynebacterium glutamicum ATCC13869 (formerly classified as Brevibacterium lactofermentum). In the present work, we investigated whether any other coryneform bacteria showed higher productivity than C. glutamicum ATCC13869. We found that most coryneform species secreted pro-transglutaminase efficiently. Moreover, we confirmed that Corynebacterium ammoniagenes ATCC6872 produced about 2.5 g/l pro-transglutaminase over a 71-h period in a jar fermentor. Our findings suggest that some other coryneform bacteria, especially C. ammoniagenes ATCC6872, are potential hosts for industrial scale protein production.     

Carbohydrate Metabolism by the Acetic Acid Bacteria

Vitamin requirements for the growth of the acetic acid bacteria were investigated extensively on a. taxonomical viewpoint and the following new findings were pointed out. Neither Acetobacter nor Intermediate strain required vitamin for the growth.

Gluconobacter required generally pantothenic acid. And some strains belonging to it did moreover somewhat of thiamine, nicotinic acid and p-aminobenzoic acid, although there was a difference of requirements between strains even in the same species. Riboflavin, pyridoxine, vitamin B₁₂, folic acid, biotin and inositol were unnecessary for the growth of the acetic acid bacteria. A taxonomical division of the acetic acid bacteria based on the vitamin requirements agreed well with that on basis of the oxidative activities for carbohydrates.     

Immunofluorescent Localization of Z-Nin in the Z-Disk

Of 14 coryneform and 2 Micrococcus strains tested, Arthrobacter globiformis IFO 12137, A. simplex IFO 12069, and Brevibacterium helvolum IFO 12073 utilized l-arginine as a sole carbon and nitrogen source, and synthesized the enzymes specific for the arginine oxygenase pathway when grown on l-arginine. The first step reaction was stimulated by FAD and aeration, and the enzyme responsible was shown to be arginine 2-monooxygenase (EC 1.13.12.1). High activities of five enzymes, including guanidinobutyramidase and ganidinobutyrase (EC 3.5.3.7), were detected in the extract of l-arginine-grown A. simplex cells. The enzymes in the last two steps, 4-aminobutyrate aminotransferase (EC 2.6.1.19) and succinate-semialdehyde dehydrogenase (EC 1.2.1.16), of B. helvolum were also induced by putrescine. These results indicate that some bacteria belonging to the coryneform group employ the arginine oxygenase pathway as a major route for l-arginine metabolism, l-arginine being degraded to succinate via 4-guanidinobutyramide and 4-guanidinobutyrate. The last part of the pathway may be common to the pathway for putrescine degradation.     

Complete reconstitution of the human coenzyme A biosynthetic pathway via comparative genomics

The biosynthesis of CoA from pantothenic acid (vitamin B5) is an essential universal pathway in prokaryotes and eukaryotes. The CoA biosynthetic genes in bacteria have all recently been identified, but their counterparts in humans and other eukaryotes remained mostly unknown. Using comparative genomics, we have identified human genes encoding the last four enzymatic steps in CoA biosynthesis: phosphopantothenoylcysteine synthetase (EC ), phosphopantothenoylcysteine decarboxylase (EC ), phosphopantetheine adenylyltransferase (EC ), and dephospho-CoA kinase (EC ). Biological functions of these human genes were verified using a complementation system in Escherichia coli based on transposon mutagenesis. The individual human enzymes were overexpressed in E. coli and purified, and the corresponding activities were experimentally verified. In addition, the entire pathway from phosphopantothenate to CoA was successfully reconstituted in vitro using a mixture of purified recombinant enzymes. Human recombinant bifunctional phosphopantetheine adenylyltransferase/dephospho-CoA kinase was kinetically characterized. This enzyme was previously suggested as a point of CoA biosynthesis regulation, and we have observed significant differences in mRNA levels of the corresponding human gene in normal and tumor cells by Northern blot analysis.     

Specialization of function among aldehyde dehydrogenases: the ALD2 and ALD3 genes are required for beta-alanine biosynthesis in Saccharomyces cerevisiae

The amino acid beta-alanine is an intermediate in pantothenic acid (vitamin B(5)) and coenzyme A (CoA) biosynthesis. In contrast to bacteria, yeast derive the beta-alanine required for pantothenic acid production via polyamine metabolism, mediated by the four SPE genes and by the FAD-dependent amine oxidase encoded by FMS1. Because amine oxidases generally produce aldehyde derivatives of amine compounds, we propose that an additional aldehyde-dehydrogenase-mediated step is required to make beta-alanine from the precursor aldehyde, 3-aminopropanal. This study presents evidence that the closely related aldehyde dehydrogenase genes ALD2 and ALD3 are required for pantothenic acid biosynthesis via conversion of 3-aminopropanal to beta-alanine in vivo. While deletion of the nuclear gene encoding the unrelated mitochondrial Ald5p resulted in an enhanced requirement for pantothenic acid pathway metabolites, we found no evidence to indicate that the Ald5p functions directly in the conversion of 3-aminopropanal to beta-alanine. Thus, in Saccharomyces cerevisiae, ALD2 and ALD3 are specialized for beta-alanine biosynthesis and are consequently involved in the cellular biosynthesis of coenzyme A.     

Distribution of membrane-bound monoamine oxidase in bacteria.

The distribution of membrane-bound monoamine oxidase in 30 strains of various bacteria was studied. Monoamine oxidase was determined by using an ammonia-selective electrode; analyses were sensitive and easy to perform. The enzyme was found in some strains of the family Enterobacteriaceae, such as Klebsiella, Enterobacter, Escherichia, Salmonella, Serratia, and Proteus. Among strains of other families of bacteria tested, only Pseudomonas aeruginosa IFO 3901, Micrococcus luteus IFO 12708, and Brevibacterium ammoniagenes IAM 1641 had monoamine oxidase activity. In all of these bacteria except B. ammoniagenes, monoamine oxidase was induced by tyramine and was highly specific for tyramine, octopamine, dopamine, and norepinephrine. The enzyme in two strains oxidized histamine or benzylamine. Correlations between the distributions of membrane-bound monoamine oxidase and arylsulfatase synthesized in the presence of tyramine were discussed.     

Distribution of membrane-bound monoamine oxidase in bacteria.

The distribution of membrane-bound monoamine oxidase in 30 strains of various bacteria was studied. Monoamine oxidase was determined by using an ammonia-selective electrode; analyses were sensitive and easy to perform. The enzyme was found in some strains of the family Enterobacteriaceae, such as Klebsiella, Enterobacter, Escherichia, Salmonella, Serratia, and Proteus. Among strains of other families of bacteria tested, only Pseudomonas aeruginosa IFO 3901, Micrococcus luteus IFO 12708, and Brevibacterium ammoniagenes IAM 1641 had monoamine oxidase activity. In all of these bacteria except B. ammoniagenes, monoamine oxidase was induced by tyramine and was highly specific for tyramine, octopamine, dopamine, and norepinephrine. The enzyme in two strains oxidized histamine or benzylamine. Correlations between the distributions of membrane-bound monoamine oxidase and arylsulfatase synthesized in the presence of tyramine were discussed.     

Energetic and carbon metabolism regulation in lysine producing brevibacterium flavum strains

Coordinated regulation of carbon and energetic metabolism enzymes is characteristic of lysine producing strains of Brevibacterium flavum. ATP is the regulating effector and changes in the stationary ATP-concentration in cells cause certain alternations of enzyme activities in the basic metabolism pathways. The goal of the experiments was the use of the biochemical regulations of methabolism in order to increase the productivity of lysine biosynthesis. Following results were received:

• Activation of TCA-cycle enzymes is compulsory for intensive lysine biosynthesis.
• The most essential of several parallel electron transport pathways in the ETC of Br. flavum is the NADH dependent, cyanid resistant, hydroxamate sensitive oxydation pathway.
• Calculations have shown, that the most economical variant is the synthesis of oxalacetate (precursor of lysine) by PEP carboxylation. Therefore strains with elevated PEP-carboxylase activity synthesize lysine in a more economical way.

     

Parameters of unbalanced growth and reversible inhibition of deoxyribonucleic acid synthesis in Brevibacterium ammoniagenes ATCC 6872 induced by depletion of Mn2+. Inhibitor studies on the reversibility of deoxyribonucleic acid synthesis

Unbalanced growth induced by depletion of manganese ions was a prerequisite for production of ribonucleotides in a high salt mineral medium with the wildtype strain Brevibacterium ammoniagenes ATCC 6872. The concentration of manganese strictly controlled the overall deoxyribonucleic acid (DNA) synthesis, whereas ribonucleic acid (RNA), protein and cell wall synthesis remained essentially unimpaired in the manganese-lacking cells.The reversibility of inhibition of overall DNA synthesis was shown by enhanced incorporation (up to threefold compared to the cultures supplied with sufficient manganese) of [8-¹⁴C] adenine into alkali-stable, trichloroacetic acid-insoluble material after subsequent addition of 10 M MnCl₂ to 15 h-old depleted cultures.The results of inhibitor studies on the restoration of overall DNA synthesis due to subsequent addition of manganese ions to depleted cultures suggest that ribonucleotide reduction is the primary target of the manganese starvation during nucleotide fermentation with Brevibacterium ammoniagenes ATCC 6872.