Purification, substrate specificity, and N-terminal amino acid sequence analysis of a beta-lactamase-free penicillin amidase from Alcaligenes sp.

A beta-lactamase-free penicillin amidase from Alcaligenes sp. active against various beta-lactams was purified to homogeneity. The enzyme can hydrolyze penicillin G to 6-amino penicillanic acid (6-APA) and furnish penicillin G from 6-APA and phenyl acetic acid by condensation. The penicillin amidase is a heterodimer of subunit masses of 63 kDa and 22 kDa, respectively. Its isoelectric point is at pH 8.5. Cephalothin was found to be the best substrate. This is a novel type II penicillin amidase which shares the properties of both type II and type III enzymes. It is thermostable and, unlike penicillin amidase from A. faecalis, its stability remains unperturbed even in presence of reductant. An inhibition study by 2-hydroxy-5-nitro benzylbromide indicated the involvement of tryptophan in catalysis by the enzyme.

Microbial transformations of chalcones: hydroxylation, O-demethylation, and cyclization to flavanones.

Microorganisms were examined for their potential to catalyze biotransformation reactions that mimic plant biosynthetic processes. Specifically, microorganisms were screened for their abilities to transform selected chalcones to flavonoid and other products. Aspergillus alliaceus UI 315 efficiently transformed 3-(2' ',3' '-dimethoxyphenyl)-1-(2'-hydroxyphenyl)propenone (2'-hydroxy-2,3-dimethoxychalcone) (1) to several products, all of which were characterized by UV, NMR, and mass spectral analyses. A. alliaceus cyclized 1 to three flavanones and to O-demethylated and hydroxylated chalcones, some of which functioned as intermediates in the cyclization process. Inhibition studies using SKF525A, metyrapone, and phenylthiocarbamide with whole cell reactions showed that as many as three cytochrome P450 enzymes may be involved in these reactions. One enzyme catalyzed chalcone cyclization; another, O-demethylation; and a third, hydroxylation of chalcones. Flavonoid products are racemic, unlike the same products that are stereoselectively cyclized in plants. This work shows that microorganisms are capable of cyclizing chalcones to form flavonoid products, thus affording a mimic of plant biosynthetic processes.

Microbial transformations of S-naproxen by Aspergillus niger ATCC 9142.

Aspergillus niger ATCC 9142 was used to catalyze the biotransformation of S(-)-naproxen (1) to three major metabolites that were isolated by solvent extraction, purified chromatographically, and characterized by mass spectrometry and NMR spectroscopy. Metabolites were identified as O-desmethylnaproxen (2), 7-hydroxynaproxen (3) and 7-hydroxy-O-desmethyinaproxen (4). The kinetics of naproxen biotransformation to 2 and 4 was established over an 84 h period to show that naproxen was completely metabolized at 36 h, the major metabolite was O-desmethylnaproxen at 24 h, and the 7-hydroxy-O-desmethylnaproxen that was formed after 24 h.

Purification and characterization of a Galactomyces reessii hydratase that converts 3-methylcrotonic acid to 3-hydroxy-3-methylbutyric acid.

Cell free extracts of Galactomyces reessii contain a hydratase as the key enzyme for the transformation of 3-methylcrotonic acid to 3-hydroxy-3-methylbutyric acid. Highest levels of hydratase activity were obtained during growth on isovaleric acid. The enzyme, an enoyl CoA hydratase, was purified 147-fold by precipitation with ammonium sulphate and successive chromatography over columns of DE-52, Blue Sepharose CL-6B and Sephacryl S-200. During purification, hydratase activity was measured spectrophotometrically (OD change at 263 nm) for 3-methylcrotonyl CoA and crotonyl CoA as substrates. The enzyme displayed highest activity with crotonyl CoA with a Kcat of 1,050,000 min(-1). The ratio of crotonyl CoA to 3-methylcrotonyl CoA activities was constant (20:1) during all steps of purification. The Kcat for crotonyl CoA was also about 20 times greater than the Kcat for 3-methylcrotonyl CoA (51,700 min(-1). The enzyme had pH and temperature optima at 7.0 and 35 degrees C, a native Mr of 260 +/- 4.5 kDa and a subunit Mr of 65 kDa, suggesting that the enzyme was a homotetramer. The pI of the purified hydratase was 5.5, and the N-terminal amino acid sequence was VPEGYAEDLLKGKMMRFFDS. Hydratase activity for 3-methylcrotonyl CoA was competitively inhibited by acetyl CoA, propionyl CoA and acetoacetyl CoA.

Microbial transformations of p-coumaric acid by Bacillus megaterium and Curvularia lunata.

p-Coumaric acid (1) is an abundant phenolic natural product that exhibits both chemoprotectant and antioxidant properties. Microbial transformation screening studies showed that 1 was converted to 4-vinylphenol (4), 4-hydroxybenzoic acid (2), caffeic acid (5), protocatechuic acid (6), and other unidentified metabolites over 144 h. 4-Vinylphenol (4) and its dimer, (2R,2S)-4-(2,3-dihydro-5-hyroxy-2-benzofuranyl) phenol (11), were produced by Bacillus megaterium, and 5-[(E)-2-carboxyethenyl]-2,3-dihydro-2-(4-hydroxyphenyl)-3-benzofurancarboxylic acid (15) and 4-hydroxybenzoic acid (2) were produced by Curvularia lunata. On the basis of deuterium-labeling experiments, B. megaterium catalyzes the nonoxidative enzymatic tautomerization of 1 to a vinylogous beta-keto acid intermediate that decarboxylates to 4. The presence of peroxidase and laccase activities in C. lunata extracts suggests that these enzymes may be involved in one-electron, p-coumaric acid dimerization in this organism.

Reactions of p-coumaric acid with nitrite: product isolation and mechanism studies.

p-Coumaric acid (1) is an abundant plant phenolic acid, a dietary chemoprotectant, and an antioxidant. The chemoprotective properties of 1 were demonstrated in vitro by its reaction with NaNO(2) in H(2)O over a range of pH values. The reaction pathway of 1 with nitrite is dependent on pH. 4-Hydroxybenzaldehyde (3, 16%), 1',4-dihydroxybenzeneacetaldehyde oxime (5, 59%), and 4-hydroxy-1'-oxo-benzeneacetaldehyde aldoxime (7, 26%) and 7-hydroxy-1,2(4H)-benzoxazin-4-one (11, 6%) were each formed at pH 2, whereas 4-(2-oxido-1,2,5-oxadiazol-3-yl)phenol (13) was formed at pH 3 (6%) and pH 7 and 10 (both 1%). Products were isolated and characterized by NMR and MS spectral analyses. Formation of benzoxazinone (11) requires the 4-phenolic functional group and the conjugated propenoic acid side chain of p-coumaric acid. The mechanism for nitrosation at pH 2 was examined by reacting 1 in H(2)(18)O/NaNO(2).

Hydroxylations and methylations of quercetin, fisetin, and catechin by Streptomyces griseus.

Preparative-scale biotransformation of quercetin (1), fisetin (7), and (+)-catechin (12) with Streptomyces griseus (ATCC 13273) resulted in the isolation and characterization of nine known hydroxylated and/or methylated (2--6, 8, 9, 11, 13a) metabolites and two previously unknown (10 and 14) metabolites. S.griseus catalyzed aromatic hydroxylations of rings A and B of quercetin and fisetin. Mono- and dimethoxy ring-B metabolites were obtained with all three substrates. Methylation appeared to occur only when catechol functional groups were present. Metabolite structures were established by FABMS, EIMS, and 1D and 2D NMR analysis.

Purification and characterization of Streptomyces griseus catechol O-methyltransferase.

A soluble (100,000 x g supernatant) methyltransferase catalyzing the transfer of the methyl group of S-adenosyl-L-methionine to catechols was present in cell extracts of Streptomyces griseus. A simple, general, and rapid catechol-based assay method was devised for enzyme purification and characterization. The enzyme was purified 141-fold by precipitation with ammonium sulfate and successive chromatography over columns of DEAE-cellulose, DEAE-Sepharose, and Sephacryl S-200. The purified cytoplasmic enzyme required 10 mM magnesium for maximal activity and was catalytically optimal at pH 7. 5 and 35 degrees C. The methyltransferase had an apparent molecular mass of 36 kDa for both the native and denatured protein, with a pI of 4.4. Novel N-terminal and internal amino acid sequences were determined as DFVLDNEGNPLENNGGYXYI and RPDFXLEPPYTGPXKARIIRYFY, respectively. For this enzyme, the K(m) for 6,7-dihydroxycoumarin was 500 +/- 21.5 microM, and that for S-adenosyl-L-methionine was 600 +/- 32.5 microM. Catechol, caffeic acid, and 4-nitrocatechol were methyltransferase substrates. Homocysteine was a competitive inhibitor of S-adenosyl-L-methionine, with a K(i) of 224 +/- 20.6 microM. Sinefungin and S-adenosylhomocysteine inhibited methylation, and the enzyme was inactivated by Hg(2+), p-chloromercuribenzoic acid, and N-ethylmaleimide.

A short formal synthesis of squalamine from a microbial metabolite.

A short formal synthesis of squalamine is described, utilizing the biotransformation product 2, which is available in one step from commercially available 3-keto-23,24-bisnorchol-4-en-22-ol (1). Regioselective C-22 oxidation and C-24 sulfation of the corresponding alcohols in the presence of a free C-7 alcohol make for an efficient preparation of squalamine intermediate 11.

Cyclic guanosine-3',5'-monophosphate and biopteridine biosynthesis in Nocardia sp.

Nocardia sp. strain NRRL 5646 contains a nitric oxide synthase (NOS) enzyme system capable of generating nitric oxide (NO) from arginine and arginine-containing peptides. To explain possible roles of the NOS system in this bacterium, guanylate cyclase (GC) and tetrahydrobiopterin (H(4)B) biosynthetic enzymes were identified in cell extracts and in culture media. Cell extracts contained GC activity, as measured by the conversion of GTP to cyclic guanosine-3',5'-monophosphate (cGMP) at 9.56 pmol of cGMP h(-1) mg of protein(-1). Concentrations of extracellular cGMP in culture media were significantly increased, from average control levels of 45 pmol cGMP liter(-1) to a maximum of 315 pmol liter(-1), in response to additions of GTP, L-arginine, H(4)B, and sodium nitroprusside to growing Nocardia cultures. On the other hand, the NOS inhibitor N(G)-nitro-L-arginine and the GC inhibitor 1H-[1,2, 4]oxadiazole[4,3-a]quinoxalin-1-one both dramatically decreased extracellular cGMP levels. Activities for GTP-cyclohydrase-1, 6-pyruvoyltetrahydropterin synthase and sepiapterin reductase, enzymes essential for H(4)B biosynthesis, were present in Nocardia culture extracts at 77.5 pmol of neopterin and 45.8 pmol of biopterin h(-1) mg of protein(-1), respectively. In Nocardia spp., as in mammals, GTP is a key intermediate in H(4)B biosynthesis, and GTP is converted to cGMP by a GC enzyme system that is activated by NO.

Biocatalytic synthesis of vanillin.

The conversions of vanillic acid and O-benzylvanillic acid to vanillin were examined by using whole cells and enzyme preparations of Nocardia sp. strain NRRL 5646. With growing cultures, vanillic acid was decarboxylated (69% yield) to guaiacol and reduced (11% yield) to vanillyl alcohol. In resting Nocardia cells in buffer, 4-O-benzylvanillic acid was converted to the corresponding alcohol product without decarboxylation. Purified Nocardia carboxylic acid reductase, an ATP and NADPH-dependent enzyme, quantitatively reduced vanillic acid to vanillin. Structures of metabolites were established by (1)H nuclear magnetic resonance and mass spectral analyses.

Abortifacient effects of a unique class of vasoactive lipids from Pinus ponderosa needles.

Pinus ponderosa needle (PN) ingestion by late pregnant cows results in decreased uterine blood flow, premature parturition, and retained placentae. Further, plasma from PN-fed cows increases caruncular arterial tone (i.e., induces prolonged contraction) in an isolated perfused bovine placentome. A novel class of vasoactive lipids was isolated and identified using a bovine placentome assay-guided fractionation of CH2Cl2 extracts of PN. Placentome perfusion tests indicated that 1-12-dodecanedioyl-dimyristate (14-12-14) was the most potent of the PN lipids for increasing caruncular arterial tone. Late pregnant guinea pigs (GP) were used to evaluate the abortifacient activity of these vasoactive lipids. In Study 1, on d 50 of gestation, part of the control diet was replaced with chopped PN (Diet A) or chopped PN subjected to sequential extraction with diethyl ether (Et2O; Diet B); Et2O and CH2Cl2 (Diet C); and Et2O, CH2Cl2, and methanol (Diet D). The GP on Diets A and B exhibited shorter (P<.01) gestation lengths and reduced (P<.01) pig birth weights than GP on the control diet or Diets C and D. Further, only GP on Diets A and B exhibited retained placentae. In Study 2, on d 50 of gestation, part of the control diet was replaced with chopped PN that had been subjected to exhaustive CH2Cl2 extraction and then infiltrated with either CH2Cl2 alone (Diet E), CH2Cl2 containing 14-12-14 (Diet F), or CH2Cl2 containing isocupressic acid (Diet G); then solvents were evaporated. The GP consuming Diet F had shorter (P<.05) gestation lengths and reduced (P<.05) pig birth weights than did GP consuming Diets E or G. The GP consuming Diet F also exhibited a high incidence of retained placentae. These data provide evidence that a unique class of vasoactive lipids in PN exhibit abortifacient activity in guinea pigs.

Novel isoflavone, cinnamic acid, and triterpenoid glycosides in soybean molasses.

Seven known isoflavones, genistein (4), daidzein (5), glycitein (6), formononetin (7), genistin (8), daidzin (9), and glycitein 7-O-beta-D-(6' '-O-acetylglucopyranoside) (10), ferulic acid, and two known saponin glycosides, soysaponin I (14) and soysaponin A2 (15), were isolated from soybean molasses. Several new compounds were also isolated and identified, including three isoflavones (1-3), two cinnamic acid ester glycosides (11) and (12), and a new saponin hexaglycoside (13). The structures of the new compounds were established on the basis of spectral data interpretation.

NMR identification of an acyl-adenylate intermediate in the aryl-aldehyde oxidoreductase catalyzed reaction.

A new one-pot synthesis was designed to prepare benzoyl-AMP under anhydrous conditions in N,N-dimethylformamide. Reaction of benzoic acid with N,N'-carbonyldiimidazole and subsequently with 5'-adenosyl monophosphate gave the mixed anhydride in 76% isolated yield. The structure of benzoyl-AMP was confirmed by mass spectroscopy and 1H-, 31P-, and 13C-NMR. The purity of the preparation was greater than 98% as indicated by 31P- and 13C-NMR. Purified aryl-aldehyde oxidoreductase was incubated in NMR tubes together with either carboxy-13C-benzoyl-AMP or carboxy-13C-benzoic acid to demonstrate that benzoyl-AMP is an active intermediate during the enzymatic reduction of benzoic acid to benzaldehyde.

Enzymatic conversion of glucose to UDP-4-keto-6-deoxyglucose in Streptomyces spp.

All of the 2,6-dideoxy sugars contained within the structure of chromomycin A3 are derived from D-glucose. Enzyme assays were used to confirm the presence of hexokinase, phosphoglucomutase, UDPG pyrophosphorylase (UDPGP), and UDPG oxidoreductase (UDPGO), all of which are involved in the pathway of glucose activation and conversion into 2,6-dideoxyhexoses during chromomycin biosynthesis. Levels of the four enzymes in Streptomyces spp. cell extracts were correlated with the production of chromomycins. The pathway of sugar activation in Streptomyces spp. involves glucose 6-phosphorylation by hexokinase, isomerization to G-1-P catalyzed by phosphoglucomutase, synthesis of UDPG catalyzed by UDPGP, and formation of UDP-4-keto-6-deoxyglucose by UDPGO.

Microbial transformations of isocupressic acid.

Microbial transformations of the labdane-diterpene isocupressic acid (1) with different microorganisms yielded several oxygenated metabolites that were isolated and characterized by MS and NMR spectroscopic analyses. Nocardia aurantia (ATCC 12674) catalyzed the cleavage of the 13,14-double bond to yield a new nor-labdane metabolite, 2. Cunninghamella elegans (-) (NRRL 1393) gave 7beta-hydroxyisocupressic acid (3) and labda-7,13(E)-diene-6beta,15, 17-triol-19-oic acid (4), and Mucor mucedo (ATCC 20094) gave 2alpha-hydroxyisocupressic acid (5) and labda-8(17),14-diene-2alpha, 13-diol-19-oic acid (6).

Gmelinosides A-L, twelve acylated iridoid glycosides from Gmelina arborea.

Besides the known iridoids 6-O-alpha-L-rhamnopyranosylcatalpol (1), 6-O-(3"-O-trans-feruloyl)-alpha-L-rhamnopyranosylcatalpol (14), 6-O-(2"-O-acetyl-3", 4"-O-di-trans-cinnamoyl)-alpha-L-rhamnopyranosylcatalpol (15) and the known phenylpropanoid glycosides verbascoside (acteoside) and martynoside, 12 new acylated iridoid glycosides named gmelinosides A-L (2-13) have been isolated from the leaves of Gmelina arborea. These compounds were structurally characterized using a variety of spectral methods.

In vitro biotransformations of isocupressic acid by cow rumen preparations: formation of agathic and dihydroagathic acids.

Isocupressic acid [15-hydroxylabda-8(17),13E-dien-19-oic acid] (1) was incubated under anaerobic conditions for 48 h in an in vitro ruminal fluid mixture and was transformed into two metabolites. The two metabolites were identified by GC/MS as agathic acid [labda-8(17),13(E)-diene-15,19-dioic acid] (4E) and dihydroagathic acid [labda-8(17)-ene-15,19-dioic acid] (6). Metabolite identities were confirmed by chemical conversions of isocupressic acid (1) and imbricataloic acid (5) into 4E and 6, respectively. Structures of synthetic metabolites were confirmed by 1H and 13C NMR, specific rotation, GC/MS, and high-resolution mass spectrometry. Plasma obtained from cows that were fed Ponderosa pine needles contained (13R,S)-dihydroagathic acid (6) but not isocupressic acid (1) or 4E. The results suggest that isocupressic acid (1) is metabolically oxidized to agathic acid (4E), subsequently reduced to (13R,S)-dihydroagathic acid (6) in the rumen, and then absorbed into the bloodstream of cattle.

Oxidations of vincristine catalyzed by peroxidase and ceruloplasmin.

The dimeric Catharanthus alkaloid vincristine (1) is oxidized to the same ring fission product in incubations with either horseradish peroxidase or the human serum copper oxidase ceruloplasmin. Horseradish peroxidase-catalyzed oxidation of vincristine requires hydrogen peroxide, whereas ceruloplasmin-catalyzed oxidation of vincristine requires chlorpromazine as a "shuttle oxidant". Preparative-scale incubations allowed for the production, isolation, structural characterization, and biological evaluation of the metabolite. The metabolite was identified as the heterocyclic ring cleavage product N-formylcatharinine (5). N-Formylcatharinine was 118 times less active than vincristine in an in vitro test against a human T-cell leukemic cell line. Therefore, these enzyme-catalyzed reactions lead to bioinactivation of vincristine.

Conversion of beta-methylbutyric acid to beta-hydroxy-beta-methylbutyric acid by Galactomyces reessii.

beta-Hydroxy-beta-methylbutyric acid (HMB) has been shown to increase strength and lean mass gains in humans undergoing resistance-exercise training. HMB is currently marketed as a calcium salt of HMB, and thus, environmentally sound and inexpensive methods of manufacture are being sought. This study investigates the microbial conversion of beta-methylbutyric acid (MBA) to HMB by cultures of Galactomyces reessii. Optimal concentrations of MBA were in the range of 5 to 20 g/liter for HMB production. Preliminary shake flask experiments indicated that HMB yields were sensitive to dissolved oxygen levels and that cell growth decreased significantly as MBA concentrations increased. Degradation of HMB was faster at acidic pH, and pH 7.0 was optimal for HMB production. Resting cells obtained from media without MBA could efficiently convert MBA to HMB. Thus, a two-step, fed-batch fermentation procedure in which biomass was first produced, followed by coaddition of MBA and glucose, while dissolved oxygen was maintained at 20% of saturation, was designed. A maximum HMB concentration of 38 g/liter was obtained after 136 h, and the molar conversion yield was more than 0.50 mol of HMB/mol of MBA during the fermentation.