Illuminating the binding interactions of galactonoamidines during the inhibition of β-galactosidase (E. coli)

Several galactonoamidines were previously identified as very potent competitive inhibitors that exhibit stabilizing hydrophobic interactions of the aglycon in the active site of β-galactosidase (Aspergillus oryzae). To elucidate the contributions of the glycon to the overall inhibition ability of the compounds, three glyconoamidine derivatives with alteration in the glycon at C-2 and C-4 were synthesized and evaluated herein. All amidines are competitive inhibitors of β-galactosidase (Escherichia coli) and show significantly reduced inhibition ability when compared to the parent. The results highlight strong hydrogen-bonding interactions between the hydroxyl group at C-2 of the amidine glycon and the active site of the enzyme. Slightly weaker H-bonds are promoted through the hydroxyl group at C-4. The inhibition constants were determined to be picomolar for the parent galactonoamidine, and nanomolar for the designed derivatives rendering all glyconoamidines very potent inhibitors of glycosidases albeit the derivatized amidines show up to 700-fold lower inhibition activity than the parent.     

Synthetic pathways of gallbladder mucosal prostanoids: the role of cyclooxygenase-1 and 2.

Acute cholecystitis is associated with increased gallbladder prostanoid formation and the inflammatory changes and prostanoid increases can be inhibited by nonsteroidal anti-inflammatory agents. Recent information indicates that prostanoids are produced by two cyclooxygenase (COX) enzymes, COX-1 and COX-2. The purpose of this study was to determine the COX enzymatic pathway in gallbladder mucosal cells involved in the production of prostanoids stimulated by inflammatory agents. Human gallbladder mucosal cells were isolated from cholecystectomy specimens and maintained in cell culture and studied in comparison with cells from a well differentiated gallbladder mucosal carcinoma cell line. COX enzymes were evaluated by Western immunoblotting and prostanoids were measured by ELISA. Unstimulated and stimulated cells were exposed to specific COX-1 and COX-2 inhibitors. In both normal and transformed cells constitutive COX-1 was evident and in gallbladder cancer cells lysophosphatidyl choline (LPC) induced the formation of constitutive COX-1 enzyme. While not detected in unstimulated normal mucosal cells and cancer cells, COX-2 protein was induced by both lipopolysaccharide (LPS) and LPC. Unstimulated gallbladder mucosal cells and cancer cells produced prostaglandin E2 (PGE2) and prostacyclin (6-keto prostaglandin F1alpha, 6-keto PGF1alpha) continuously. In freshly isolated normal gallbladder mucosal cells, continuously produced 6 keto PGF1alpha was inhibited by both COX-1 and COX-2 inhibitors while PGE2 levels were not affected. Both LPS and LPC stimulated PGE2 and 6 keto PGF1alpha formation were blocked by COX-2 inhibitors in freshly isolated, normal human gallbladder mucosal cells and in the gallbladder cancer cells. The prostanoid response of gallbladder cells stimulated by proinflammatory agents is inhibited by COX-2 inhibitors suggesting that these agents may be effective in treating the pain and inflammation of gallbladder disease.     

Partial characterization of the cytosol 3 alpha-hydroxysteroid: NAD(P)+oxidoreductase of rat ventral prostate.

The cytosol 3alpha-hydroxysteroid dehydrogenase of rat ventral prostate has been partially purified. The rates of both the oxidation and reduction by crude and partially purified enzymes have been measured with a variety of radioactive substrates, and the effects of several inhibitor steroids have been assessed. Four conclusions have been drawn from the study. First, no detectable 3beta-androstanediol was formed from dihydrotestosterone, and the oxidation of 3beta-androstanediol was undetectable. Second, the cytosol enzyme exhibits a distinct and unique substrate specificity in that steroids with keto or hydroxyl substitution on the 11th carbon of the steroid cannot serve as substrates or as inhibitors of the enzyme. Third, either 5alpha or 5 beta reduction of delta4,3-keto steroids must take place before the steroids can serve as substrates of the enzyme. Fourth, many delta4,3-keto steroids that cannot act as substrates for the enzyme inhibit the enzyme competitively and may well serve as physiological regulators of the reaction in intact cell.     

Baker's yeast mediated reduction of β-keto esters and β-keto amides in an organic solvent system

The baker's yeast mediated reduction of four β-keto esters in petroleum ether indicated that the size of the group attached to the keto carbon affected their reactivity. Ethyl 3-phenyl-3-oxopropanoate (1), which has a phenyl group directly attached to the keto carbon, is incompletely reduced using 20 g yeast/mmol substrate, ethyl 4-phenyl-3-oxobutanoate (2), which has one methylene group between the phenyl and keto carbon, was also incompletely reduced using 20 g yeast/mmol, although the extent of reduction was about double that of (1), ethyl 5-phenyl-3-oxopentanoate (3), which has two methylene groups between the phenyl and keto carbon, is completely reduced using 10 g yeast/mmol and ethyl 3-oxobutanoate (4), which has a methyl group attached to the keto carbon shows complete reduction using only 1 g yeast/mmol. The corresponding β-keto amides are considerably less reactive than the corresponding β-keto esters with only the amides derived from ethyl 3-oxobutanoate indicating any significant reduction using 20 g yeast/mmol.     

Identification of P-glycoprotein mutations causing a loss of steroid recognition and transport.

P-glycoproteins transport a wide variety of hydrophobic compounds out of cells. While the diversity of transported molecules suggests a mechanism involving broad specificity, there is evidence of significant discrimination within given classes of molecules. One example of this behavior is transport of corticosteroids by the murine mdr1 P-glycoprotein. The presence of hydroxyl groups, associated with specific steroid carbon atoms, regulates the ability of corticosteroids to be transported. This specificity is demonstrated here by experiments measuring the ability of steroids to inhibit drug transport. The results indicate that a keto oxygen associated with the 3- and 20-carbon atoms, as well as a 17-carbon hydroxyl group, each acts to enhance steroidal P-glycoprotein inhibitory activity. Moreover, inhibitory steroids can be used for directed selection of variant cells, expressing mutated P-glycoproteins with a severely impaired ability to transport dexamethasone. The five mutations, reported here, are located within transmembrane domains 4-6, proximal to the cytoplasmic interface. The altered P-glycoproteins exhibit reduced capacity to be inhibited by specific steroids, suggesting decreased capacity to bind these molecules avidly. Studies comparing the relative inhibitory activity of a series of steroids indicate that these mutations alter recognition of the 17alpha-hydroxyl group and the 20-keto oxygen atom.     

Hydroxyl radical generation by the tetracycline antibiotics with free radical damage to DNA, lipids and carbohydrate in the presence of iron and copper salts

Tetracycline antibiotics caused the degradation of carbohydrate in the presence of a ferric salt at pH 7.4. This degradation appeared to involve hydroxyl radicals since the damage was substantially reduced by the presence of catalase, superoxide dismutase, scavengers of the hydroxyl radical and metal chelators. Similarly, the tetracycline antibiotics in the presence of a ferric salt greatly stimulated the peroxidation of liposomal membranes. This damage, which did not implicate the hydroxyl radical, was significantly reduced by the addition of chain-breaking antioxidants and metal chelators. Only copper salts in the presence of tetracycline antibiotics, however, caused substantial damage to linear duplex DNA. Studies with inhibitors suggested that damage to DNA did involve hydroxyl radicals.     

Hydrixyl radical generation by the tetracycline antibiotics with free radical damage to DNA, lipids and carbohydrate in the presence of iron and copper salts

Tetracycline antibiotics caused the degradation of carbohydrate in the presence of a ferric salt at pH 7.4. This degradation appeared to involve hydroxyl radicals since the damage was substantially reduced by the presence of catalase, superoxide dismutase, scavengers of the hydroxyl radical and metal chelators. Similarly, the tetracycline antibiotics in the presence of a ferric salt greatly stimulated the peroxidation of liposomal membranes. This damage, which did not implicate the hydroxyl radical, was significantly reduced by the addition of chain-breaking antioxidants and metal chelators. Only copper salts in the presence of tetracycline antibiotics, however, caused substantial damage to linear duplex DNA. Studies with inhibitors suggested that damage to DNA did involve hydroxyl radicals.     

Mutagenicity of plant flavonoids: structural requirements for mutagenic activity in Salmonella typhimurium.

40 compounds structurally related to the plant flavonol quercetin were tested for mutagenic activity in Salmonella typhimurium strain TA98. 10 flavonols, quercetin, myricetin, rhamnetin, galangin, kaempferol, tamarixetin, morin, 3'-O-methylquercetin, 7,4'-di-O-methylquercetin and 5,7-di-O-methyl-quercetin, exhibited unequivocal mutagenic activity. 4 compounds, quercetin, myricetin, rhamnetin and 5,7-di-O-methylquercetin, were active without metabolic activation, although metabolic activation markedly enhanced their activity. All 4 have free hydroxyl groups at the 3' and 4' positions of the B ring. The other active compounds required an in vitro rat-liver metabolizing system for significant activity. Structural features which appear essential for mutagenic activity in this strain are a basic flavanoid ring structure with (1) a free hydroxyl group at the 3 position, (2) a double bond at the 2, 3 position, (3) a keto group at the 4 position, and (4) a structure which permits the proton of the 3-hydroxyl group to tautomerise to a 3-keto compound. The data are consistent with the requirement for a B ring structure that permits oxidation to quininoid intermediates. Free hydroxyl groups in the B ring are not essential for activity if a rat-liver metabolic activating system is employed. Data from 12 compounds which differ only at the essential sites described above indicate that the structural requirements for mutagenicity in strain TA100 are the same as those for activity in strain TA98. Based on the above structural requirements, a metabolic pathway for flavonol activation to DNA-reactive species is proposed.     

Evidence that the 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD1) is regulated by pentose pathway flux. Studies in rat adipocytes and microsomes

11 beta-hydroxysteroid dehydrogenase type 1 (11 beta-HSD1) catalyzes the interconversion of biologically inactive 11 keto derivatives (cortisone, 11-dehydrocorticosterone) to active glucocorticoids (cortisol, corticosterone) in fat, liver, and other tissues. It is located in the intraluminal compartment of the endoplasmic reticulum. Inasmuch as an oxo-reductase requires NADPH, we reasoned that 11 beta-HSD1 would be metabolically interconnected with the cytosolic pentose pathway because this pathway is the primary producer of reduced cellular pyridine nucleotides. To test this theory, 11 beta-HSD1 activity and pentose pathway were simultaneously measured in isolated intact rodent adipocytes. Established inhibitors of NAPDH production via the pentose pathway (dehydroandrostenedione or norepinephrine) inhibited 11 beta-HSD1 oxo-reductase while decreasing cellular NADPH content. Conversely these compounds slightly augmented the reverse, or dehydrogenase, reaction of 11 beta-HSD1. Importantly, using isolated intact microsomes, the inhibitors did not directly alter the tandem microsomal 11 beta-HSD1 and hexose-6-phosphate dehydrogenase enzyme unit. Metabolites of 11 beta-HSD1 (corticosterone or 11-dehydrocorticosterone) inhibited or increased pentose flux, respectively, demonstrating metabolic interconnectivity. Using isolated intact liver or fat microsomes, glucose-6 phosphate stimulated 11 beta-HSD1 oxo-reductase, and this effect was blocked by selective inhibitors of glucose-6-phosphate transport. In summary, we have demonstrated a metabolic interconnection between pentose pathway and 11 beta-HSD1 oxo-reductase activities that is dependent on cytosolic NADPH production. These observations link cytosolic carbohydrate flux with paracrine glucocorticoid formation. The clinical relevance of these findings may be germane to the regulation of paracrine glucocorticoid formation in disturbed nutritional states such as obesity.     

Prostacyclin-independence in beta-adrenoceptor mediated renin release from dog renal cortical slices

There is some controversy regarding whether stimulation of renin release by the beta-adrenergic system is dependent on prostaglandin (PG) production. We have examined this problem in renal cortical slices of the dog and have obtained the following results: (1) Isoproterenol (4 X 10(-6) M) stimulated renin release, but had no effect on the formation of 6-keto PGF1 alpha, a stable metabolite of PGI2; (2) Indomethacin (2 X 10(-5) M) had no effect on isoproterenol stimulated renin release, but inhibited 6-keto PGF1 alpha formation; (3) Dibutyryl cyclic AMP (10(-3) M) stimulated both renin release, and 6-keto PGF1 alpha release. Indomethacin (2 X 10(-5) M) did not inhibit dibutyryl cyclic AMP-stimulated renin release, but did inhibit the production of 6 keto PGF1 alpha. These results indicate that the beta-adrenoceptor mediated renin release does not depend on the formation of PGI2, but renin release is dependent on cyclic AMP formation.     

Rerouting Citrate Metabolism in Lactococcus lactis to Citrate-Driven Transamination

Oxaloacetate is an intermediate of the citrate fermentation pathway that accumulates in the cytoplasm of Lactococcus lactis ILCitM(pFL3) at a high concentration due to the inactivation of oxaloacetate decarboxylase. An excess of toxic oxaloacetate is excreted into the medium in exchange for citrate by the citrate transporter CitP (A. M. Pudlik and J. S. Lolkema, J. Bacteriol. 193:4049-4056, 2011). In this study, transamination of amino acids with oxaloacetate as the keto donor is described as an additional mechanism to relieve toxic stress. Redirection of the citrate metabolic pathway into the transamination route in the presence of the branched-chain amino acids Ile, Leu, and Val; the aromatic amino acids Phe, Trp, and Tyr; and Met resulted in the formation of aspartate and the corresponding α-keto acids. Cells grown in the presence of citrate showed 3.5 to 7 times higher transaminase activity in the cytoplasm than cells grown in the absence of citrate. The study demonstrates that transaminases of L. lactis accept oxaloacetate as a keto donor. A significant fraction of 2-keto-4-methylthiobutyrate formed from methionine by citrate-driven transamination in vivo was further metabolized, yielding the cheese aroma compounds 2-hydroxy-4-methylthiobutyrate and methyl-3-methylthiopropionate. Reducing equivalents required for the former compound were produced in the citrate fermentation pathway as NADH. Similarly, phenylpyruvate, the transamination product of phenylalanine, was reduced to phenyllactate, while the dehydrogenase activity was not observed for the branched-chain keto acids. Both α-keto acids and α-hydroxy acids are known substrates of CitP and may be excreted from the cell in exchange for citrate or oxaloacetate.     

3′and 4′ and equatorial amino and hydroxy derivatives of neamine

All eight of the possible amino and hydroxyl derivatives at the 3′ and 4′ positions of neamine were prepared. The compounds are either 3′-deoxy or 4′-deoxy analogs having axial or equatorial amino or hydroxyl groups at the other position. The position and configuration of the hydroxyl groups from the reduction of the two 3′, 4′-epoxides, 11 and 12, were established. The 3′-keto and 4′-keto derivatives (26 and 23) and also the corresponding oximino acetates, 33b and 35b, were reduced and the structures of the products determined. Hydrolytic cleavage of the exo-methylene epoxides at the 3′ and 4′ positions gave the diols, 47 and 45. N.m.r. data for the 3′ and 4′ acetates and acetamides are presented.     

Hydrogen bonding in lignin: a Fourier transform infrared model compound study

Hydrogen bonding plays an important role in the thermal and mechanical properties of biopolymers. To investigate hydrogen bond formation in lignin, an abundant natural polymer found in plants, Fourier transform infrared (FTIR) analysis of various lignin model compounds was performed. Four monomeric model compounds and one dimeric model compound were studied under various conditions. FTIR analysis revealed aliphatic hydroxyl groups form stronger hydrogen bonds than phenolic hydroxyl groups. Further, the dimeric biphenyl-type structure formed significantly stronger intermolecular hydrogen bonds as compared to the other monomeric model compounds. Results from the model compound studies were used to explain the observed complex hydrogen-bonding system present in both softwood and hardwood technical lignins. Together with chemical analysis, we discuss the difference in hydrogen bonding between hardwood and softwood lignin and the observed differences in the glass transition temperature.     

O-trimethylsilylquinoxalinol derivatives of aromatic α-keto acids : Mass spectra and quantitative gas chromatography

As an extension of earlier work on aliphatic α-keto acids, a method is described for the quantitative gas chromatographic determination of urinary aromatic α-keto acids. The keto acids are derivatized with o-phenylenediamine to yield the quinoxalinols. These compounds are chromatographed after trimethylsilyation.The aromatic keto acids are stabilized by sodium dithionite (4 mg/ml urine) and storage below 0°. The final derivatives are stable for weeks at room temperature.Low resolution mass spectra are reported. The fragmentation mechanims are elucidated by analysis of O-trimethylsilyl-(TMS)-quinoxalinols, O-(TMS-d₉)-quinoxalinols and O-TMS-6(7)-chloroquinoxalinols.     

In vitro analysis of iron chelating activity of flavonoids

Flavonoids have been demonstrated to possess miscellaneous health benefits which are, at least partly, associated with iron chelation. In this in vitro study, 26 flavonoids from different subclasses were analyzed for their iron chelating activity and stability of the formed complexes in four patho/physiologically relevant pH conditions (4.5, 5.5, 6.8, and 7.5) and compared with clinically used iron chelator deferoxamine. The study demonstrated that the most effective iron binding site of flavonoids represents 6,7-dihydroxy structure. This site is incorporated in baicalein structure which formed, similarly to deferoxamine, the complexes with iron in the stoichiometry 1:1 and was not inferior in all tested pH to deferoxamine. The 3-hydroxy-4-keto conformation together with 2,3-double bond and the catecholic B ring were associated with a substantial iron chelation although the latter did not play an essential role at more acidic conditions. In agreement, quercetin and myricetin possessing all three structural requirements were similarly active to baicalein or deferoxamine at the neutral conditions, but were clearly less active in lower pH. The 5-hydroxy-4-keto site was less efficient and the complexes of iron in this site were not stable at the acidic conditions. Isolated keto, hydroxyl, methoxyl groups or an ortho methoxy-hydroxy groups were not associated with iron chelation at all.     

Biocatalytic asymmetric amination of carbonyl functional groups – a synthetic biology approach to organic chemistry

Transaminases catalyze amino transfer reactions from amino donors such as amino acids or amines to keto acids or ketones to give chiral amino acid or amines in optically pure form. α-Amino acid dehydrogenases catalyze the asymmetric reductive amination of α-keto acids using ammonia as amino donor to furnish L -amino acids. The distinct features and synthetic application of these two enzymes are reviewed in an effort to illustrate their promising and challenging aspects in serving as approaches to the direct asymmetric synthesis of optically pure amines from the corresponding keto compounds, a formidable problem in organic chemistry.     

Determination of the structure of tetrahedral transition state analogues bound at the active site of chymotrypsin using 18O and 2H isotope shifts in the 13C NMR spectra of glyoxal inhibitors

The peptide-derived glyoxal inhibitor Z-Ala-Pro-Phe-glyoxal, where Z is benzyloxycarbonyl, is an extremely potent inhibitor of chymotrypsin. When it is bound to chymotrypsin both the glyoxal (RCOCHO) keto and aldehyde carbons are sp3 hybridized with chemical shifts of 100.7 and 91.4 ppm, respectively. However it is has not been shown whether these carbons are bound as hydrates or whether the active-site serine has reacted with them to form the corresponding hemiketal or hemiacetal. In this study we use 18O isotope shifts to determine whether one or two exchangeable oxygen atoms are attached to the glyoxal keto or aldehyde carbons when it is free in water or bound to alpha-chymotrypsin. Both the 18O isotope shifts at the free and enzyme-bound aldehyde carbons were approximately 0.04 ppm showing that it is hydrated in both the free and bound forms. The 18O isotope shift for the free hydrated keto carbon at 96.6 ppm was 0.046-0.049 ppm, but this was reduced to 0.026 ppm when the glyoxal inhibitor was bound to alpha-chymotrypsin showing that the nonexchangeable serine hydroxyl group has formed a hemiketal with glyoxal keto carbon. Deuterium isotope shifts on the 13C NMR signals from the glyoxal inhibitor when it free and hydrated, when it is bound to chymotrypsin, as well as when it forms a model hemiketal confirm that the serine hydroxyl group has formed a hemiketal with the glyoxal keto carbon. The reasons for the different reaction specificities of glyoxal inhibitors for the active-site nucleophiles of serine and cysteine proteases are discussed.     

Purification and regulatory properties of chicken heart prostaglandin E 9-ketoreductase.

Prostaglandin E 9-ketoreductase was purified from chicken heart by ammonium sulfate fractionation, and DEAE-Sephadex, hydroxylapatite and phosphocellulose chromatography. Two peaks of activity were resolved during the phosphocellulose chromatographic step. Both peaks were stimulated by a substance that was not bound to the phosphocellulose column. This stimulatory substance was destroyed by treatment with phosphodiesterase and 0.1 M NaOH. It was heat-stable (100 degrees, 2 min), nondialyzable, and resistant to treatment with pronase, ribonuclease, and deoxyribonuclease; but it was dialyzable after heating or digestion with pronase. Sodium pyrophosphate also enhanced the activities of the prostaglandin E 9-ketoreductases as did angiotensin I; but not angiotensin II. In the presence of 3':5'-cyclic AMP, AMP, or several other ribonucleotides, the enhancing effects of the natural stimulatory substance, sodium pyrophosphate or angiotensin I were blocked, but these ribonucleotides themselves had little effect on the enzymes activity. The substrate specificities of the two prostaglandin E 9-ketoreductases were also studied. Both the 9-keto group and the 15-keto group of 15-ketoprostaglandin F2 alpha could be converted to the corresponding hydroxyl group; the 15-keto group was reduced faster than the 9-keto group. Prostaglandin D2, a prostaglandin with a 9-hydroxyl and an 11-keto group, could not be converted to prostaglandin F2 alpha nor could cyclohexanone be converted to cyclohexanol by the prostaglandin E 9-ketoreductase.     

Measurement of prostaglandin F2α metabolites by radioimmunoassay

Antibodies against 15 keto PGF_2α and 13,14 dihydro 15 keto PGF_2α were produced in goats and rabbits using the appropriate prostaglandin protein conjugate. Tritium labeled 15-keto, and 13,14 dihydro 15-keto PGF_2α were prepared from ³H-PGF_2α. These antibodies and ³H-labeled compounds were used to develop radioimmunoassays for the respective F_2α metabolites. The antibodies had relatively little cross-reactivity (≤0.1%) with the parent F_2α molecule. Infusion of PGF_2α in monkeys increased 15-keto-h₂ levels 10–20 fold higher than PGF_2α in peripheral plasma. The levels of this metabolite were not altered detectably during clotting, indicating relatively slow rates of PGF_2α metabolism in vitro. These assays should be useful to follow release rates of exogenous prostaglandins from various formulations and delivery systems, and in vivo tissue synthesis of PGF_2α, where low levels preclude measuring the parent compound.