Su-Il Kang

Flavonoids biotransformation by bacterial non-heme dioxygenases, biphenyl and naphthalene dioxygenase

This review details recent progresses in the flavonoid biotransformation by bacterial non-heme dioxygenases, biphenyl dioxygenase (BDO), and naphthalene dioxygenase (NDO), which can initially activate biphenyl and naphthalene with insertion of dioxygen in stereospecfic and regiospecific manners. Flavone, isoflavone, flavanone, and isoflavanol were biotransformed by BDO from Pseudomonas pseudoalcaligenes KF707 and NDO from Pseudomonas sp. strain NCIB9816-4, respectively. In general, BDO showed wide range of substrate spectrum and produced the oxidized products, whereas NDO only metabolized flat two-dimensional substrates of flavone and isoflavone. Furthermore, biotransformation of B-ring skewed substrates, flavanone and isoflavanol, by BDO produced the epoxide products, instead of dihydrodiols. These results support the idea that substrate-driven reactivity alteration of the Fe-oxo active species may occur in the active site of non-heme dioxygenases. The study of flavonoid biotransformation by structurally-well defined BDO and NDO will provide the substrate structure and reactivity relationships and eventually establish the production of non-plant-originated flavonoids by means of microbial biotechnology.

Identification of fungal metabolites of anticonvulsant drug carbamazepine.

Carbamazepine, which has been used in the treatments of epilepsy, is often found in the environment. Although metabolism of carbamazepine by humans and rats has been characterized, the environmental fate of carbamazepine has not been studied. In this study, two model fungi Cunninghamella elegans ATCC 9245 and Umbelopsis ramanniana R-56, which have previously shown diverse metabolic activities, were tested for metabolism of carbamazepine. Both fungi produced three metabolites each (C1–C3 and M1–M3). All six metabolites showed [M + H]+ at m/z 253, suggesting addition of one oxygen to the parent compound. High-performance liquid chromatography and liquid chromatography–mass spectrometric analysis detected 10, 11-dihydro-10, 11-epoxycarbamazepine as a major product (C3 (47%) and M3 (85%)) and 3-hydroxycarbamazepine (C2 (15%) and M2 (7%)) from carbamazepine through mixed mono-oxidation reactions in both fungal strains. C. elegans was confirmed to produce 2-hydroxycarbamazepine (C1 (38%)) while U. ramanniana produced a yet unidentified ring-hydroxylated metabolite (M1 (8%)). The current study suggests that carbamazepine is likely to be subjected to initially diverse mono-oxygenation reactions by fungal metabolisms, resulting in the formation of the corresponding metabolites, which were similarly found in mammalian metabolisms.

Location of flavone B-ring controls regioselectivity and stereoselectivity of naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4

Naphthalene dioxygenase (NDO) from Pseudomonas sp. strain NCIB 9816-4 incorporated dioxygen at the C7 and C8 positions on the A-rings of flavone and isoflavone with different stereoselectivity, resulting in the formation of (7S,8S)-dihydroxy-2-phenyl-7,8-dihydro-4H-chromen-4-one (flavone-cis-(7S,8S)-dihydrodiol) and (7R,8R)-dihydroxy-3-phenyl-7,8-dihydro-4H-chromen-4-one (isoflavone-cis-(7R,8R)-dihydrodiol), respectively. In addition, NDO was shown to incorporate dioxygen at the C5 and C6 positions on the A-ring and the C2′ and C3′ positions on the B-ring of isoflavone, resulting in the production of (5S,6R)-dihydroxy-3-phenyl-5,6-dihydro-4H-chromen-4-one (isoflavone-cis-(5S,6R)-dihydrodiol) and 3-[(5S,6R)-5,6-dihydroxycyclohexa-1,3-dienyl]-4H-chromen-4-one (isoflavone-cis-(2′R,3′S)-dihydrodiol), respectively. The metabolites were identified by LC/MS, 1H, and 13C NMR analyses and TD-SCF calculations combined with CD spectroscopy. In the case of flavone biotransformation, formation of flavone-(7S,8S)-dihydrodiol is likely to be the result of hydrogen bond interactions between the substrate and the active site of the dioxygenase. On the contrary, regioselective dioxygenation of isoflavone was found not to occur, and this may be due to the fact that the same hydrogen bonds that occur in the case of the flavone reaction cannot be established due to steric hindrance caused by the position of the B-ring. It is therefore proposed that the regioselectivity and stereoselectivity of NDO from strain NCIB 9816-4 are controlled by the position of the phenyl ring on flavone molecules.

Catalytic mechanism of inulinase from Arthrobacter sp. S37.

Detailed catalytic roles of the conserved Glu323, Asp460, and Glu519 of Arthrobacter sp. S37 inulinase (EnIA), a member of the glycoside hydrolase family 32, were investigated by site-directed mutagenesis and pH-dependence studies of the enzyme efficiency and homology modeling were carried out for EnIA and for D460E mutant. The enzyme efficiency (k(cat)/K(m)) of the E323A and E519A mutants was significantly lower than that of the wild-type due to a substantial decrease in k(cat), but not due to variations in K(m), consistent with their putative roles as nucleophile and acid/base catalyst, respectively. The D460A mutant was totally inactive, whereas the D460E and D460N mutants were active to some extent, revealing Asp460 as a catalytic residue and demonstrating that the presence of a carboxylate group in this position is a prerequisite for catalysis. The pH-dependence studies indicated that the pK(a) of the acid/base catalyst decreased from 9.2 for the wild-type enzyme to 7.0 for the D460E mutant, implicating Asp460 as the residue that interacts with the acid/base catalyst Glu519 and elevates its pK(a). Homology modeling and molecular dynamics simulation of the wild-type enzyme and the D460E mutant shed light on the structural roles of Glu323, Asp460, and Glu519 in the catalytic activity of the enzyme.

Absolute configurations of isoflavan-4-ol stereoisomers

Isoflavan-4-ol has been synthesized quantitatively from the reduction of isoflavone in the presence of Pd/C and ammonium formate under N(2) atmosphere. Isolation of cis- and trans-isomers was achieved by flash column chromatography and each enantiomer was separated by Sumi-Chiral column chromatography. Absolute configurations of four stereoisomers were determined by circular dichroism spectroscopy.

Absolute configuration-dependent epoxide formation from isoflavan-4-ol stereoisomers by biphenyl dioxygenase of Pseudomonas pseudoalcaligenes strain KF707

Biphenyl dioxygenase from Pseudomonas pseudoalcaligenes strain KF707 expressed in Escherichia coli was found to exhibit monooxygenase activity toward four stereoisomers of isoflavan-4-ol. LC–MS and LC–NMR analyses of the metabolites revealed that the corresponding epoxides formed between C2′ and C3′ on the B-ring of each isoflavan-4-ol substrate were the sole products. The relative reactivity of the stereoisomers was found to be in the order: (3S,4S)-cis-isoflavan-4-ol > (3R,4S)-trans-isoflavan-4-ol > (3S,4R)-trans-isoflavan-4-ol > (3R,4R)-cis-isoflavan-4-ol and this likely depended upon the absolute configuration of the 4-OH group on the isoflavanols, as explained by an enzyme–substrate docking study. The epoxides produced from isoflavan-4-ols by P. pseudoalcaligenes strain KF707 were further abiotically transformed into pterocarpan, the molecular structure of which is commonly found as part of plant-protective phytoalexins, such as maackiain from Cicer arietinum and medicarpin from Medicago sativa.

Assessment of the Occurrence and Risks of Emerging Organic Pollutants (EOPs) in Ikpa River Basin Freshwater Ecosystem, Niger Delta-Nigeria

Abstract The levels of some emerging organic pollutants (EOPs) including endocrine disrupting compounds, pharmaceuticals and personal care products were quantified in surface water of a freshwater ecosystem, the Ikpa River Basin, Nigeria using liquid chromatography/high resolution tandem mass spectrometry (LC–MS/MS). In addition, leachates and storm water samples collected from nearby dumpsites were also analysed to assess the effect on water quality. Seventeen compounds were detected at the nanogramme-per-litre levels and the ecological risks of selected compounds assessed based on predicted no-effect concentrations derived from comparison of toxicity data recorded for green algae, fish and invertebrate with the maximum measured environmental concentrations, to obtain risk quotients. Some of the compounds showed some level of widespread occurrence or persistence. Also, bisphenol A, chloramphenicol, erythromycin, triclocarban and triclosan were the most important EOPs detected in the study area that may pose detrimental effects to the aquatic organisms based on the outcome of the risk assessment.

Distribution of Toxin Genes and Enterotoxins in Bacillus thuringiensis Isolated from Microbial Insecticide Products.

Bacillus thuringiensis microbial insecticide products have been applied worldwide. Although a few cases of B. thuringiensis foodborne illness have been reported, little is known about the toxigenic properties of B. thuringiensis isolates. The aims of this study were to estimate the pathogenic potential of B. thuringiensis selected from microbial insecticide products, based on its possession of toxin genes and production of enterotoxins. Fifty-two B. thuringiensis strains selected from four kinds of microbial insecticide products were analyzed. PCR assay for detection of toxin genes and immunoassay for detection of enterotoxins were performed. The hemolysin BL complex as a major enterotoxin was produced by 17 (32.7%), whereas the nonhemolytic enterotoxin complex was detected in 1 (1.9%) of 52 B. thuringiensis strains. However, cytK, entFM, and ces genes were not detected in any of the tested B. thuringiensis strains. The potential risk of food poisoning by B. thuringiensis along with concerns over B. thuringiensis microbial insecticide products has gained attention recently. Thus, microbial insecticide products based on B. thuringiensis should be carefully controlled.