Microbial Production of Ursodeoxycholic Acid from Lithocholic Acid by Fusarium equiseti M41

A fungus identified as Fusarium equiseti was isolated from soil and found to carry out 7β-hydroxylation of lithocholic acid to ursodeoxycholic acid (35% yield; 350 mg/liter) in 112 h.     

Optimum conditions for ursodeoxycholic acid production from lithocholic acid by Fusarium equiseti M41.

Ursodeoxycholic acid dissolves cholesterol gallstones in humans. In the present study optimum conditions for ursodeoxycholic acid production by Fusarium equiseti M41 were studied. Resting mycelia of F. equiseti M41 showed maximum conversion at 28 degrees C, pH 8.0, and dissolved oxygen tension of higher than 60% saturation. Monovalent cations, such as Na+, K+, and Rb+, stimulated the conversion rate more than twofold. In the presence of 0.5 M KCl, the initial uptake rate and equilibrium concentration of lithocholic acid (substrate) were enhanced by 5.7- and 1.7-fold, respectively. We confirmed that enzyme activity catalyzing 7 beta-hydroxylation of lithocholic acid was induced by substrate lithocholic acid. The activity in the mycelium was controlled by dissolved oxygen tension during cultivation: with a dissolved oxygen tension of 15% and over, the activity peak appeared at 25 h of cultivation, whereas the peak was delayed to 34 and 50 h with 5 and 0% dissolved oxygen tension, respectively. After reaching the maximum, the 7 beta-hydroxylation activity in the mycelium declined rapidly at pH 7.0, but the decline was retarded by increasing the pH to 8.0. Several combinations of operations, such as pH shift (from pH 7 to 8), addition of 0.5 M KCl, and dissolved oxygen control, were applied to the production of ursodeoxycholic acid in a jar fermentor, and a much larger amount of ursodeoxycholic acid (1.2 g/liter) was produced within 96 h of cultivation.     

Optimum conditions for ursodeoxycholic acid production from lithocholic acid by Fusarium equiseti M41

Ursodeoxycholic acid dissolves cholesterol gallstones in humans. In the present study optimum conditions for ursodeoxycholic acid production by Fusarium equiseti M41 were studied. Resting mycelia of F. equiseti M41 showed maximum conversion at 28 degrees C, pH 8.0, and dissolved oxygen tension of higher than 60% saturation. Monovalent cations, such as Na+, K+, and Rb+, stimulated the conversion rate more than twofold. In the presence of 0.5 M KCl, the initial uptake rate and equilibrium concentration of lithocholic acid (substrate) were enhanced by 5.7- and 1.7-fold, respectively. We confirmed that enzyme activity catalyzing 7 beta-hydroxylation of lithocholic acid was induced by substrate lithocholic acid. The activity in the mycelium was controlled by dissolved oxygen tension during cultivation: with a dissolved oxygen tension of 15% and over, the activity peak appeared at 25 h of cultivation, whereas the peak was delayed to 34 and 50 h with 5 and 0% dissolved oxygen tension, respectively. After reaching the maximum, the 7 beta-hydroxylation activity in the mycelium declined rapidly at pH 7.0, but the decline was retarded by increasing the pH to 8.0. Several combinations of operations, such as pH shift (from pH 7 to 8), addition of 0.5 M KCl, and dissolved oxygen control, were applied to the production of ursodeoxycholic acid in a jar fermentor, and a much larger amount of ursodeoxycholic acid (1.2 g/liter) was produced within 96 h of cultivation.     

6α-Hydroxylation of taurochenodeoxycholic acid and lithocholic acid by CYP3A4 in human liver microsomes

The aim of the present study was to identify the enzymes in human liver catalyzing hydroxylations of bile acids. Fourteen recombinant expressed cytochrome P450 (CYP) enzymes, human liver microsomes from different donors, and selective cytochrome P450 inhibitors were used to study the hydroxylation of taurochenodeoxycholic acid and lithocholic acid. Recombinant expressed CYP3A4 was the only enzyme that was active towards these bile acids and the enzyme catalyzed an efficient 6α-hydroxylation of both taurochenodeoxycholic acid and lithocholic acid. The V_max for 6α-hydroxylation of taurochenodeoxycholic acid by CYP3A4 was 18.2 nmol/nmol P450/min and the apparent K_m was 90 μM. Cytochrome b₅ was required for maximal activity. Human liver microsomes from 10 different donors, in which different P450 marker activities had been determined, were separately incubated with taurochenodeoxycholic acid and lithocholic acid. A strong correlation was found between 6α-hydroxylation of taurochenodeoxycholic acid, CYP3A levels (r²=0.97) and testosterone 6β-hydroxylation (r²=0.9). There was also a strong correlation between 6α-hydroxylation of lithocholic acid, CYP3A levels and testosterone 6β-hydroxylation (r²=0.7). Troleandomycin, a selective inhibitor of CYP3A enzymes, inhibited 6α-hydroxylation of taurochenodeoxycholic acid almost completely at a 10 μM concentration. Other inhibitors, such as α-naphthoflavone, sulfaphenazole and tranylcypromine had very little or no effect on the activity. The apparent K_m for 6α-hydroxylation of taurochenodeoxycholic by human liver microsomes was high (716 μM). This might give an explanation for the limited formation of 6α-hydroxylated bile acids in healthy humans. From the present results, it can be concluded that CYP3A4 is active in the 6α-hydroxylation of both taurochenodeoxycholic acid and lithocholic acid in human liver.     

Acetone and Butanol Production by Clostridium acetobutylicum in a Synthetic Medium

The effect of the component concentrations of a synthetic medium on acetone and butanol fermentation by Clostridium acetobutylicum ATCC 824 was investigated. Cell growth was dependent on the presence of Mg, Fe, and K in the medium. Mg and Mn had deleterious effects when in excess. Ammonium acetate in excess caused acid fermentation. The metabolism was composed of two phases: an acid phase and a solvent one. Low concentrations of glucose allowed the first phase only. The theoretical ratio of the conversion of glucose to solvents, which was 28 to 33%, was obtained with the following medium: MgSO₄, 50 to 200 mg/liter; MnSO₄, 0 to 20 mg/liter; KCl, 0.015 to 8 g/liter (an equivalent concentration of K⁺ was supplied in the form of KH₂PO₄ and K₂HPO₄); FeSO₄, 1 to 50 mg/liter; ammonium acetate, 1.1 to 2.2 g/liter; para-aminobenzoic acid, 1 mg/liter; biotin, 0.01 mg/liter; glucose, 20 to 60 g/liter.     

Sulphydryl groups in the maintenance of the activity of binding protein for N-1-naphthylphthalamic acid fromAcer pseudoplatanus cells

Binding protein for N-1-naphthylphthalamic acid (NPA), an auxin transport inhibitor, was studied by analysis of the effects of reactions which modify particular amino acid side chains upon their binding activity. Na₂SO₃, N-ethylmaleimide (NEM) and dithiobisnitrobenzoic acid all inhibited the specific binding of NPA to its binding protein fromAcer pseudoplatanus L. cells. The presence of 10^-6 M Na₂SO₃ in the binding assay reduced the affinity of the binding protein to NPA from K_d of 1.5 ￡ 10^-8 M to K_d of 2.1 ￡ 10_-8 M, while concentration of the binding protein was not significantly changed. When the same analysis was applied to NPA binding to the NEM-treated membrane particles, it was found that NEM inactivated binding without changing the affinity for NPA. This study revealed the importance of sulphydryl group(s) in the maintenance of NPA binding protein activity.     

The use of aurovertin to determine the F1 content of submitochondrial particles and the ATPase complex

(1) The concentration of aurovertin-binding sites calculated from fluorimetric titrations of submitochondrial particles is equal to the F₁ concentration, calculated from the concentration of F₁-binding sites in stripped particles.(2) Direct binding experiments show that the fluorescence enhancement of aurovertin bound to submitochondrial particles and the isolated ATPase complex is less (or absent) at higher concentrations than at lower concentrations. The binding data can be described by ‘specific’ and ‘non-specific’ binding. The concentration of the ‘specific’ sites is twice that derived from fluorimetric titrations.(3) After dissociation of the bound F₁ with LiCl, fluorimetric titrations with aurovertin yield linear Scatchard plots. The fluorescence enhancement and K_D are equal to those of the β-subunit-aurovertin complex. The concentration of β-subunits is double the concentration of F₁.(4) It is concluded that both for submitochondrial particles and the isolated ATPase complex the most reliable and simple way to determine the F₁ content is to dissociate the F₁ with LiCl, spin down the insoluble material and titrate the supernatant (containing free β-subunit) with aurovertin.     

Chemical synthesis of bile acid acyl-adenylates and formation by a rat liver microsomal fraction

In mammals, unconjugated bile acids formed in the intestine by bacterial deconjugation are reconjugated (N-acylamidated) with taurine or glycine during hepatocyte transport. Activation of the carboxyl group of bile acids to form acyl-adenylates is a likely key intermediate step in bile acid N-acylamidation. To gain more insight into the process of bile acid adenylate formation, we first synthesized the adenylates of five common, natural bile acids (cholic, deoxycholic, chenodeoxycholic, ursodeoxycholic, and lithocholic acid), and confirmed their structure by proton NMR. We then investigated adenylate formation by subcellular fractions of rat liver (microsomes, mitochondria, cytosol) using a newly developed LC method for quantifying adenylate formation. The highest activity was observed in the microsomal fraction. The reaction required Mg²⁺ and its optimum pH was about pH 7.0. In term of maximum velocity (V_max) and the Michaelis constant (K_m), the catalytic efficiency of the enzyme under the conditions used was highest with cholic acid of the bile acids tested. The formation of cholyl-adenylate was strongly inhibited by lithocholic and deoxycholic acid, as well as by palmitic acid; ibuprofen and valproic acid were weak inhibitors. In cholestatic disease, such adenylate formation might lead to subsequent bile acid conjugation with glutathione or proteins.     

Relationship of prolactin receptors to concanavalin A binding

Concanavalin A, which binds to specific carbohydrate determinants on the cell surface, was used to investigate the binding of prolactin to its receptors in liver membranes from female rats. The binding of ¹²⁵I-labeled ovine prolactin to receptors was sharply inhibited by concanavalin A. This effect was reversed by the competitive sugar α-methyl-D-mannopyranoside and thus required the presence of specifically bound lectin. Concentrations of concanavalin A of up to 50 μg/ml caused a progressive decrease in the apparent affinity of the prolactin receptor for hormone. When higher concentrations were used, the number of available binding sites decreased. Concanavalin A-resistant receptors, about 30% of the total, had the same dissociation constant (K_d) as the controls. The binding of ¹²⁵I-labeled concanavalin A in the same membrane preparations showed the presence of two distinct types of concanavalin A binding. At low concentrations, the lectin bound with high affinity (K_d ≈ 6.6 · 10^?8 M). At high lectin concentrations, low affinity (K_d ≈ 6.7 · 10^?5 M) binding predominated. Since high affinity concanavalin A binding was saturated at 50 μg/ml, this class of binding most likely alters the affinity of the prolactin receptor for hormone; low affinity concanavalin A binding may mask prolactin receptors, making them inaccessible to the hormone.Binding sites for concanavalin A and prolactin appear to be independent but closely related since (i) concanavalin A did not displace bound prolactin from its receptor, and (ii) detergent-solubilized ¹²⁵I-labeled prolactin-receptor complexes bound to concanavalin A-Sepharose and were eluted by α-methyl-D-mannopyranoside.     

Genome-Based Metabolic Engineering of Mannheimia succiniciproducens for Succinic Acid Production

Succinic acid is a four-carbon dicarboxylic acid produced as one of the fermentation products of anaerobic metabolism. Based on the complete genome sequence of a capnophilic succinic acid-producing rumen bacterium, Mannheimia succiniciproducens, gene knockout studies were carried out to understand its anaerobic fermentative metabolism and consequently to develop a metabolically engineered strain capable of producing succinic acid without by-product formation. Among three different CO₂-fixing metabolic reactions catalyzed by phosphoenolpyruvate (PEP) carboxykinase, PEP carboxylase, and malic enzyme, PEP carboxykinase was the most important for the anaerobic growth of M. succiniciproducens and succinic acid production. Oxaloacetate formed by carboxylation of PEP was found to be converted to succinic acid by three sequential reactions catalyzed by malate dehydrogenase, fumarase, and fumarate reductase. Major metabolic pathways leading to by-product formation were successfully removed by disrupting the ldhA, pflB, pta, and ackA genes. This metabolically engineered LPK7 strain was able to produce 13.4 g/liter of succinic acid from 20 g/liter glucose with little or no formation of acetic, formic, and lactic acids, resulting in a succinic acid yield of 0.97 mol succinic acid per mol glucose. Fed-batch culture of M. succiniciproducens LPK7 with intermittent glucose feeding allowed the production of 52.4 g/liter of succinic acid, with a succinic acid yield of 1.16 mol succinic acid per mol glucose and a succinic acid productivity of 1.8 g/liter/h, which should be useful for industrial production of succinic acid.     

A novel binding site for bile acids on human serum albumin

Binding sites of bile acids on human serum albumin were studied using various probes: dansylsarcosine (site I probe), 7-anilinocoumarin-4-acetic acid (ACAA, site II probe), 5-dimethylaminonaphthelene-1-sulfonamide (DNSA, site III probe), cis-parinaric acid (probe for fatty acid binding site) and bilirubin. Bile acids competitively inhibited the binding of dansylsarcosine to human serum album whereas bile acids enhanced the binding of ACAA, DNSA, cis-parinaric acid and bilirubin. Considering the concentrations of bile acids required to inhibit the binding of dansylsarcosine to human serum albumin, the secondary binding site of bile acids may correspond to site I. Dissociation constants (K_d) of the primary binding sites of lithocholic and chenodeoxycholic acid to human serum albumin were approximately 0.2 and 4 μM, respectively, which was measured by equilibrium dialysis at 37° C. All the bile acids and their sulfates and glucuronides inhibited the binding of chenodeoxycholic acid to human serum albumin. Lithocholic and chenodeoxycholic acid and their sulfates and glucuronides exhibited more inhibition than cholic acid and its conjugates. In conclusion, bile acids may bind to a novel binding site on human serum albumin.     

In-vitro auxin binding to particulate cell fractions from corn coleoptiles

Summary When low concentrations (e.g. 10^-6 M) of labelled 3-indoleacetic acid (¹⁴C-IAA) or -naphthaleneacetic acid (¹⁴C-NAA) are added in vitro to homogenates of corn coleoptiles, radioactivity is reversibly bound to pelletable particles. From the saturation kinetics of the binding it is possible to estimate an apparent K _M between 10^-6 M and 10^-5 M and a concentration of specific sites of 10^-7–10^-6 M per tissue volume.The binding is auxin-specific. Among many compounds tested, only auxins and such auxin analogues that are known to interact directly with auxin in transport and/or growth were found to interfere with this binding. For instance, the growth-active d-dichlorophenoxyisopropionic acid at 10^-4 M inhibits ¹⁴C-NAA binding more than the less active l-isomer.The auxin-binding fractions are practically free of DNA and cytochrome-C oxidase and contain binding sites for 1-naphthylphthalamic acid. The results are discussed in context with the hyothesis—derived mainly from physiological data—that auxin receptors are localized at the plasma membrane.     

Synthesis and proteasome inhibition of lithocholic acid derivatives

A new class of proteasome inhibitors was synthesized using lithocholic acid as a scaffold. Modification at the C-3 position of lithocholic acid with a series of acid acyl groups yielded compounds with a range of potency on proteasome inhibition. Among them, the phenylene diacetic acid hemiester derivative (13) displayed the most potent proteasome inhibition with IC₅₀ = 1.9 μM. Enzyme kinetic analysis indicates that these lithocholic acid derivatives are noncompetitive inhibitors of the proteasome.     

Specific binding of phorbol ester tumor promoters to mouse skin

The tumor promoter 20-³H-phorbol 12,13-dibutyrate bound in a specific manner to particulate preparations from both whole mouse skin and mouse epidermis. The binding, which was comparable in both whole skin and epidermal preparations, occurred rapidly, was reversible upon addition of non-radioactive ligand and showed high affinity (K_D = 2.4 × 10^?8 M). The potencies of phorbol esters for inhibiting binding of ³H-PDBu corresponded to their biological and tumor-promoting activities: phorbol 12-myristate 13-acetate, K_I = 0.74 nM; phorbol 12,13-didecanoate, K_I = 16 nM; phorbol 12,13-dibenzoate, K_I = 82 nM; mezerein, K_I = 98 nM; phorbol 12,13-diacetate, K_I = 3 μM; phorbol 12,13,20-triacetate, K_I = 5.6 μM; phorbol 13-acetate, K_I = 64 μM. The biologically inactive derivatives phorbol (0.88 mM) and 4α-phorbol 12,13-didecanoate (15 μM) did not inhibit binding. Likewise, ³H-PDBu binding was only weakly inhibited by phorbol-related diterpenes which are highly inflammatory but nonpromoting. These structure-activity relationships suggest that the ³H-PDBu binding activity mediates phorbol ester tumor promotion. ³H-PDBu binding was not inhibited by the nonphorbol promoters examined. Similarly, it was not blocked by compounds which antagonize (dexamethasone acetate, 2 μM; retinoic acid, 10 μM) or mimic (epidermal growth factor, 100 ng/ml; melittin, 25 μg/ml; PGE₂, 1 μM) some of the effects of the phorbol esters in vivo or in vitro.     

Chymotrypsin inhibitor from potatoes: interaction with target enzymes

The interactions of chymotrypsin, subtilisin and trypsin with a low MW proteinase inhibitor from potatoes were investigated. The K_i value calculated for the binding of inhibitor to chymotrypsin was 1.6 ± 0.9 × 10^?10M, while the second-order rate constant for association was 6 × 10⁵ M^?1/sec. Although binding was not observed to chymotrypsin which had been treated with diisopropyl fluorophosphate or with l-tosylamide-2-phenylethyl chloromethyl ketone, the 3-methylhistidine-57 derivative bound inhibitor with a K_i value of 9.6 × 10^?9 M. The inhibitor also exhibited a tight association with subtilisin (K_i < 4 × 10^?9 M). In contrast, little inhibition of trypsin was observed, and this was believed to be due to low levels of a contaminant in our preparations. No evidence for reactive site cleavage was observed after incubation of the inhibitor with catalytic amounts of chymotrypsin or subtilisin at acid pH.     

Levorphanol inhibition of Ca++ binding to synaptic membranes in vitro

Levorphanol inhibits Ca⁺⁺ binding to synaptic membranes in a dose dependent fashion (0.5–10nM) and induces release of calcium previously bound to membranes. The inhibition is noncompetitive and exhibits a K_i of 9 × 10^?9M. Naloxone (10 nM) blocks this inhibition while dextrophan (100nM) is without effects. The Ca⁺⁺ binding exhibits a high affinity (K_D=0.84μM) and a saturable capacity of 350 picomles/mg protein. The binding curve is sigmoid with a Hill coefficient of 2.2, while in the presence of levorphanol (0.5 to 50nM), the curve becomes hyperbolic with a Hill coefficient of 1.1. The data suggest the calcium and opiate receptor binding sites are in close proximity and may be functionally linked through sub-unit interactions.     

The use of 8-azido-ATP and 8-azido-ADP as photoaffinity labels of the ATP synthase in submitochondrial particles: Evidence for a mechanism of ATP hydrolysis involving two independent catalytic sites?

8-Azido-ATP is a substrate for the ATP synthase in submitochondrial particles with a V_max equal to 6% of the V_max with ATP. The K_m values for 8-azido-ATP are similar to those for ATP. ATP synthase in submitochondrial particles can bind maximally 2 mol 8-N-ATP or 8-N-ADP per mole and the inhibition of ATP hydrolysis by covalently bound N-ATP or N-ADP is proportional to the saturation of the enzyme with inhibitor, similar to the results obtained with isolated F₁. Both 8-N-ATP and 8-N-ADP are bound mainly to the β subunits and at all levels of saturation the distribution of the label is 77% to the β and 23% to the α subunits. It is proposed that the binding of 8-azido-AXP itself is mainly to the β subunit, but that part of the nitreno radicals formed during excitation with light reacts with an amino acid of the α subunit, due to the location of the binding site at an interface between a β and an α subunit. Partial saturation with 8-N-ATP, under conditions that the concentration of 8-azido-ATP during the incubation is intermediate between the low and high K_m values, does not abolish the apparent negative cooperativity of ATP hydrolysis. It is concluded that this apparent cooperativity is not due to the presence of two different catalytic sites, nor to a cooperativity between the two catalytic sites, but to interaction between the catalytic sites and regulatory sites.     

Inorganic pyrophosphatase and nucleoside diphosphatase in the parasitic protozoon, Entamoeba histolytica.

Entamoeba histolytica contains two acid pyrophosphatases. One is an inorganic pyrophosphatase with a relatively high K_m ( ? 1 mM) and no cation requirement. The other is a nucleoside diphosphatase with a relatively low K_m ( ? 50 μM) and Ca²⁺ requirement. No Mg²⁺ dependent neutral or alkaline inorganic pyrophosphatase is present. The pyrophosphatases are localized in subcellular particles, display structure-linked latency and are tightly bound to membranes.     

Dissimilation of Carbon Monoxide to Acetic Acid by Glucose-Limited Cultures of Clostridium thermoaceticum

Clostridium thermoaceticum was cultivated in glucose-limited media, and the dissimilation of CO to acetic acid was evaluated. We found that cultures catalyzed the rapid dissimilation of CO to acetic acid and CO₂, with the stoichiometry obtained for conversion approximating that predicted from the following reaction: 4CO + 2H₂O → CH₃CO₂H + 2CO₂. Growing cultures formed approximately 50 mmol (3 g) of CO-derived acetic acid per liter of culture, with the rate of maximal consumption approximating 9.1 mmol of CO consumed/h per liter of culture. In contrast, resting cells were found not to dissimilate CO to acetic acid. ¹⁴CO was incorporated, with equal distribution between the carboxyl and methyl carbons of acetic acid when the initial cultivation gas phase was 100% CO, whereas ¹⁴CO₂ preferentially entered the carboxyl carbon when the initial gas phase was 100% CO₂. Significantly, in the presence of saturating levels of CO, ¹⁴CO₂ preferentially entered the methyl carbon, whereas saturating levels of CO₂ yielded ¹⁴CO-derived labeling predominantly in the carboxyl carbon. These findings are discussed in relation to the path of carbon flow to acetic acid.