David S. Fukuda

Microbial transformation of A23187, a divalent cation ionophore antibiotic.

A23187 is an ionophore antibiotic that forms dimeric complexes with divalent cations such an Mn2+ and Ca2+. Over 200 randomly selected soil microorganisms were incubated with A23187. None of these cultures was capable of transforming this compound. In contrast, many microorganisms were able to modify the methyl ester of A23187. The transformation products produced by one culture, Streptomyces chartreusis, were isolated and identified as 16-hydroxy-N-demethyl A23187 methyl ester, 16-hydroxy-A23187 methyl ester, and N-demethyl A23187 methyl ester. These ester derivatives lack most of the ionophore properties of the acids and cannot readily form dimeric complexes with divalent cations. However, they could be hydrolyzed by a mild treatment with ethanolic KOH to free acids that possess good ionophore activity. The use of the ester substrate in conjunction with the hydrolysis procedure is, at the present time, the only known method for microbiologically producing A23187 derivatives.

Studies on the mechanism of action of A80915A, a semi-naphthoquinone natural product, as an inhibitor of gastric (H+-K+)-ATPase

A semi-naphthoquinone natural product, A80915A, produced by Streptomyces aculeolatus was found to be a potent inhibitor of gastric (H(+)-K+)-ATPase, the enzyme responsible for acid secretion in the stomach. Enzyme activity was measured by potassium-stimulated hydrolysis of ATP or p-nitrophenolphosphate with enzyme prepared from the stomach fundic mucosa of pigs. Concentration-dependent inhibition was observed with an IC50 of about 2-3 microM for both ATPase and p-nitrophenylphosphatase. A Hill plot indicated that the enzyme has two binding sites for A80915A. Inhibition was not affected by the presence of the reducing agent dithiothreitol, indicating a lack of involvement of enzyme sulfhydryl groups. A 30-min incubation of enzyme with increasing drug concentrations followed by a 10-fold dilution did not alter the IC50, indicating that A80915A does not covalently modify the enzyme. Coincubation of enzyme with 3.8 microM A80915A resulted in time-dependent inhibition. The rate of inhibition was slowed significantly by the presence of 20 mM potassium, rubidium and ammonium but not by 20 mM sodium, lithium and choline, or by 40 mM sucrose. The level of inhibition was influenced by the order of addition of potassium and drug to the enzyme. Taken together, these studies indicate that inhibition by A80915A is dependent on the conformation of gastric (H(+)-K+)-ATPase and that potassium slows the rate of inhibition by converting the enzyme to a conformation where the drug binding site is not as accessible. The mode of action of A80915A is distinct from that of two well characterized proton pump inhibitors, omeprazole and SCH 28080.

A82775b and a82775c, novel metabolites of an unknown fungus of the order sphaeropsidales

Compounds A82775B (1) and A82775C (2) were isolated as non-antimicrobial by-products during attempts to isolate an elusive G+, G- antibiotic from culture A82775. an unidentified mold. A82775 factors B and C are present in both the mycelium and filtered broth. The metabolites were recovered from the filtered broth by adsorption on Diaion HP-20 nonfunctionalized macroreticular resin and purified by preparative reverse phase HPLC on C18 bonded phase supports. The structure elucidation of A82775 factors B (1) and C (2) has been accomplished by spectroscopic means including COSY, NOESY, INAPT, HMQC and 2D-INADEQUATE NMR experiments.

Microbiological transformation of cannabinoids.

Microorganisms were screened for their ability to modify 2 synthetic cannabinoid substrates (I andII). Structure analyses revealed that microorganisms transformed the substrates by (a) primary oxidation of the side chain, β-oxidation of the side chain, ketone formation on the side chain or cyclohexene ring, (b) secondary hydroxylation on the side chain, (c) aromatization of the cyclohexene ring, and (d) tertiary hydroxylation at the b/c ring juncture.

Cephalosporin acetylesterase (Bacillus subtilis).

Publisher Summary This chapter discusses the assay procedure and properties of cephalosporin acetylesterase (Bacillus subtilis). Cephalosporin esterase activity is readily assayed by titration with an automatic pH star. The reaction generates acetic acid that causes a drop in pH of the reaction mixture. A standardized KOH solution is automatically added to maintain the pH at a preset value (usually pH = 7.0). The amount of KOH added per unit of time is directly proportional to the reaction rate. The rate of KOH addition is automatically recorded on a moving chart, and the initial reaction rate can be determined from the slope of the line. The enzyme is extremely stable. It may be stored in an unbuffered solution at 25° and pH - 7.0 for 3 weeks with little or no loss of activity. In addition to cephalosporins, the enzyme will hydrolyze mono- and triacetin, α-naphthyl acetate, and glucose pentaacetate. The enzyme does not hydrolyze casein, acetanilide, p-nitrophenylacetate, p-nitrophenylsulfate, and it is not inhibited by arsenilic acid or bis (p-nitrophenyl) phosphate.

Preparation and Properties of a Cephalosporin Acetylesterase Adsorbed onto Bentonite

A cephalosporin acetylesterase produced by Bacillus subtilis was immobilized by adsorption onto bentonite. The immobilized enzyme (EI) and the soluble enzyme (ES) exhibited Michaelis-Menton kinetics with 7-aminocephalosporanic acid (7-ACA): Km = 2.8 × 10−3 M and Km = 3.2 × 10−3 M, respectively. Similar kinetics were observed with 7-(thiophene-2-acetamido)cephalosporanic acid (cephalothin), but the Km value measured with EI (3.7 × 10−3 M) was less than one-half that measured with this substrate and ES. The reduction in Km value was correlated with the ability of bentonite to adsorb cephalothin. The reaction products, acetate and deacetyl-7-ACA, were weak competitive inhibitors of ES and EI. The Ki values for EI were 5.0 × 10−2 M for acetate and 3.6 × 10−2 M for deacetyl-7-ACA. Similar values were measured with ES and these substrates. EI retained about 80% of its initial activity after 3 weeks of storage in solution at 25 C. However, the enzyme dissociated from the bentonite particles during the deacetylation reaction. This dissociation was minimized by cross-linking EI with glutaraldehyde or bis-dimethyladipimidate, or by adding Al(OH)3 to the suspension. With the latter addition, EI was stabilized so that it could be reused nine times before one-half of the initial activity was lost.

Enzymatic removal of a cephalosporin methyl ester blocking group

Abstract Over 7000 microorganisms were screened to find an enzyme source for the hydrolysis of a C 4 methyl ester blocking group on 7-aminodesacetoxycephalosporanic acid (7-ADCA). Only one culture, Streptomyces capillispira Mertz and Higgens nov. sp., produced an enzyme that catalysed the reaction. Enzyme synthesis in a defined mineral salts medium was repressed by NH 3 and amino acids. Under optimum fermentation conditions, the maximum rate of substrate hydrolysis was 6 × 10 −10 mol min −1 mg −1 cell. The enzyme was recovered from the mycelia and partially purified by gel filtration. Kinetic studies by pH-stat titration indicated that the pH optimum was 7.5–8.5, the temperature optimum was 25–30°C, and the substrate K m value was 2.3 mg ml −1 . The reaction products, 7-ADCA and methanol, were weak competitive inhibitors of the enzyme with K 1 values of 6.63 and 0.188 mg ml −1 , respectively. The enzyme also hydrolysed cefaclor and cephalexin methyl esters but did not hydrolyse cephalosporin ethyl esters. With further improvements in enzyme yields and stability, enzymatic deblocking of cephalosporins could provide an alternative to chemical deblocking processes.