Jaehong Han

Oxygen-independent decarbonylation of aldehydes by cyanobacterial aldehyde decarbonylase: a new reaction of diiron enzymes.

The search for new biofuels has generated increased interest in biochemical pathways that produce hydrocarbons.[1] Although hydrocarbons are simple molecules, the biosynthesis of molecules that lack any chemical functional groups is surprisingly challenging.[2] Biochemical reactions that remove functionality, such as decarboxylations, dehydrations and reduction of double bonds, invariably rely on the presence of adjacent functional groups to stabilize unfavorable transition states. Enzymes involved in hydrocarbon biosynthesis are therefore of interest both for applications in biofuels production and because of the unusual and chemically difficult reactions they catalyze.[3] One enzyme that has attracted particular interest, is aldehyde decarbonylase (AD), which catalyzes the decarbonylation of long-chain fatty aldehydes, to the corresponding alkanes.[4]

Oxygen-independent alkane formation by non-heme iron-dependent cyanobacterial aldehyde decarbonylase: Investigation of kinetics and requirement for an external electron donor

Cyanobacterial aldehyde decarbonylase (cAD) is, structurally, a member of the di-iron carboxylate family of oxygenases. We previously reported that cAD from Prochlorococcus marinus catalyzes the unusual hydrolysis of aldehydes to produce alkanes and formate in a reaction that requires an external reducing system but does not require oxygen [Das et al. (2011) Angew. Chem. 50, 7148-7152]. Here we demonstrate that cADs from divergent cyanobacterial classes, including the enzyme from N. puntiformes that was reported to be oxygen dependent, catalyze aldehyde decarbonylation at a much faster rate under anaerobic conditions and that the oxygen in formate derives from water. The very low activity (<1 turnover/h) of cAD appears to result from inhibition by the ferredoxin reducing system used in the assay and the low solubility of the substrate. Replacing ferredoxin with the electron mediator phenazine methosulfate allowed the enzyme to function with various chemical reductants, with NADH giving the highest activity. NADH is not consumed during turnover, in accord with the proposed catalytic role for the reducing system in the reaction. With octadecanal, a burst phase of product formation, k(prod) = 3.4 ± 0.5 min(-1), is observed, indicating that chemistry is not rate-determining under the conditions of the assay. With the more soluble substrate, heptanal, k(cat) = 0.17 ± 0.01 min(-1) and no burst phase is observed, suggesting that a chemical step is limiting in the reaction of this substrate.

Stereospecific Biotransformation of Dihydrodaidzein into (3S)-Equol by the Human Intestinal Bacterium Eggerthella Strain Julong 732

ABSTRACT Stereochemical course of isoflavanone dihydrodaidzein (DHD) reduction into the isoflavan (3S)-equol via tetrahydrodaidzein (THD) by the human intestinal anaerobic bacterium Eggerthella strain Julong 732 was studied. THD was synthesized by catalytic hydrogenation, and each stereoisomer was separated by chiral high-performance liquid chromatography. Circular dichroism spectroscopy was used to elucidate the absolute configurations of four synthetic THD stereoisomers. Rapid racemization of DHD catalyzed by Julong 732 prevented the substrate stereospecificity in the conversion of DHD into THD from being confirmed. The absolute configuration of THD, prepared by reduction of DHD in the cell-free incubation, was assigned as (3R,4S) via comparison of the retention time to that of the authentic THD by chiral chromatography. Dehydroequol (DE) was unable to produce the (3S)-equol both in the cell-free reaction and in the bacterial transformation, negating the possible intermediacy of DE. Finally, the intermediate (3R,4S)-THD was reduced into (3S)-equol by the whole cell, indicating the inversion of stereochemistry at C-3 during the reduction. A possible mechanism accounting for the racemization of DHD and the inversion of configuration of THD during reduction into (3S)-equol is proposed.

Stereospecific microbial production of isoflavanones from isoflavones and isoflavone glucosides.

A Gram-negative anaerobic microorganism, MRG-1, isolated from human intestine showed high activities of deglycosylation and reduction of daidzin, based on rapid TLC analysis. A rod-shaped strain MRG-1was identified as a new species showing 91.0% homology to Coprobacillus species, based on 16S rRNA sequence analysis. The strain MRG-1 showed β-glucosidase activity toward daidzin and genistin, and daidzein and genistein were produced, respectively. However, the strain MRG-1 did not react with flavone glycosides, flavanone glycosides, and isoflavone C-glucoside. Besides, MRG-1 showed stereoselective reductase activity to isoflavone, daidzein, genistein, 7-hydroxyisoflavone, and formononetin, resulting in the formation of corresponding R-isoflavanone enantiomers. The new isoflavanones of 7-hydroxyisoflavanone and dihydroformononetin were characterized by NMR, and the absolute configurations of the enantiomers were determined with CD spectroscopy. The kinetic study of the anaerobic biotransformation showed both activities were exceptionally fast compared to the reported conversion by other anaerobic bacteria.

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.

Conversion of (3S,4R)-Tetrahydrodaidzein to (3S)-Equol by THD Reductase: Proposed Mechanism Involving a Radical Intermediate

To elucidate the mechanism of (3S)-equol biosynthesis, (2,3,4-d(3))-trans-THD was synthesized and converted to (3S)-equol by THD reductase in Eggerthella strain Julong 732. The position of the deuterium atoms in (3S)-equol was determined by (1)H NMR and (2)H NMR spectroscopy, and the product was identified as (2,3,4(alpha)-d(3))-(3S)-equol. All the deuterium atoms were retained, while the OH group at C-4 was replaced by a hydrogen atom with retention of configuration. To explain the deuterium retention in this stereospecific reduction, we propose a mechanism involving radical intermediates.

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.

Absolute configuration determination of isoflavan-4-ol stereoisomers

Elucidation of the correct stereochemistry of the metabolite is essential for the mechanistic study of bioactive compounds. Isoflavan-4-ol has the same chiropical chromophore as THD, the biosynthetic precursor of the potent phytoestrogen S-equol. Interested in the correct absolute configuration of isoflavan-4-ol stereoisomers and to compare the available practical approaches for the absolute configuration determination, complete absolute configuration analysis of isoflavan-4-ol stereoisomers has been carried out with by means of ECD and VCD spectroscopy as well as modified Mosher method. Theoretical TD-DFT computations resulted in a poor simulation of the observed experimental ECD spectra, and thus inconclusive absolute configuration assignments of isoflavan-4-ol stereoisomers were obtained. However, DFT-assisted VCD spectroscopic analyses successfully determined correct absolute configurations, and further confirmed by modified Mosher method.

Deglycosylation of isoflavone C-glycosides by newly isolated human intestinal bacteria

BACKGROUND Plant isoflavones are mostly present in the glycoside form. Isoflavone aglycones produced by intestinal microflora are reported to be more bioactive than the glycoside form. However, the deglycosylation of isoflavone C-glycosides is known to be rare, and is less studied. RESULTS Three new bacteria were isolated from human faecal samples, two of which hydrolysed the C-glycosidic bond of puerarin, daidzein-8-C-glucoside. They were identified as two Lactococcus species, herein designated as MRG-IFC-1 and MRG-IFC-3, and an Enterococcus species, herein designated MRG-IFC-2, based on their 16S rDNA sequences. From a reactivity study, it was found that Lactococcus sp. MRG-IFC-1 and Enterococcus sp. MRG-IFC-2 hydrolysed isoflavone C- and O-glycosides, as well as the flavone O-glycoside apigetrin, but could not hydrolyse the flavone C-glycosidic bond of vitexin. The other Lactococcus sp., MRG-IF-3, could not hydrolyse the C-glycosidic linkage of puerarin, while it showed a broad substrate spectrum of O-glycosidase activity similar to the other two bacteria. Puerarin was completely converted to daidzein within 100 min by Lactococcus sp. MRG-IFC-1 and Enterococcus sp. MRG-IFC-2, which is the fastest conversion among the reported human intestinal bacteria. CONCLUSION Two new puerarin-metabolising human intestinal bacteria were isolated and identified, and the deglycosylation activity for various flavonoid glycosides was investigated. The results could facilitate the study of C-glycosidase reaction mechanisms, as well as the pharmacokinetics of bioactive C-glycoside natural products.

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.