Kei Shimoda

Biotransformation of enones with biocatalysts : two enone reductases from Astasia longa

Abstract The stereochemistry and mechanism in the reduction of the C–C double bond of carvone by the cultured cells of Astasia longa , a nonchlorophyllous cell line classified in Euglenales , was studied. The reduction of the C–C double bond of carvone with the cultured cells involved the anti -addition of hydrogen atom from the si face at the α-position and the re face at the β-position of carbonyl group. Two different enone reductases were isolated from the cultured cells of A. longa . Both reductases catalyzed stereospecifically the anti -addition of hydrogen atoms from the si face at C-1 and the re face at C-6. However, one of the reductases participated in a hydrogen transfer of the pro -4 R hydrogen of NADH to C-6 position of carvone and the other used the pro -4 S hydrogen of NADH.

Novel reductase participation in the syn-addition of hydrogen to the CC bond of enones in the cultured cells of Nicotiana tabacum

A reductase isolated from cultured cells of Nicotiana tabacum has been characterized and used in the reduction of a CC bond adjacent to a carbonyl group. The stereochemistry of the latter reaction has been investigated by 2H NMR and mass spectroscopy. It was found that the reductase reduces stereospecifically the CC bond of verbenone and carvone by syn addition of hydrogen from the re face at the β-position and the re face at the α-position to the carbonyl group; the hydrogen atoms participating in the enzymatic reduction at the α- and β-positions originate from the medium (H2O) and the pro-4S hydrogen of NADPH, respectively.

Introduction of oxygenated functional groups into 3-carene and 2-pinene by cultured cells.

The biotransformation of the monoterpene hydrocarbons 3-carene and 2-pinene by cell suspension cultures of Nicotiana tabacum and Catharanthus roseus was investigated. The cultures have the ability to regio- and enantioselectively introduce the oxygenated functional groups into the C = C double bond and the allylic positions of the substrates.

Biotransformation of phenolic compounds by the cultured cells of Catharanthus roseus

Abstract The cultured cells of Catharanthus roseus were able to convert 2-, 3-, and 4-hydroxybenzyl alcohols into their corresponding hydroxybenzyl-β- d -glucopyranosides or β- d -glucopyranosylbenzyl alcohols, and then convert 2- and 3-hydroxybenzyl-β- d -glucopyranosides into primeverosides and vicianosides. Further, the C. roseus cells were capable of hydroxylation of 2-hydroxybenzoic acid to afford 2,5-dihydroxybenzoic acid and then glucosylation of the newly introduced phenolic hydroxyl group.

Stereochemistry in the reduction of enones by the reductase from euglena gracilis z

A reductase, which catalyses the NADH-dependent reduction of the C=C bond adjacent to the carbonyl group, was characterized with regard to the stereochemistry of the hydrogen transfer into the substrate. The reductase was isolated from Euglena gracilis Z and was found to reduce stereospecifically the C=C bond of carvone by anti-addition of hydrogen from the si face at α-position to the carbonyl group and the re face at β-position. The hydrogen atoms participating in the enzymatic reduction at α- and β-position to the carbonyl group originate from the medium and the pro-4R hydrogen of NADH, respectively.

A 38 kDa allylic alcohol dehydrogenase from the cultured cells of Nicotiana tabacum.

An NADP+-dependent alcohol dehydrogenase (allyl-ADH) was isolated from the cultured cells of Nicotiana tabacum. The allyl-ADH was found to be efficient for the dehydrogenation of secondary allylic alcohols rather than saturated secondary alcohols and it was specific for the S-stereoisomer of the alcohols. The enzyme catalyzed the reversible reaction whereby the carbonyl group of enones is reduced to the corresponding allylic alcohol or vice versa. Two possible primary structures of the allyl-ADH were deduced by the sequence analyses of full-length cDNAs (allyl-ADH1 and ally-ADH2), which were cloned by the PCR method. These analyses indicated that the allyl-ADHs are composed of 343 amino acids having the molecular weights 38083 and 37994, respectively, and they showed approximately 70% homology to the NADP+-dependent oxidoreductases belonging to a plant zeta-crystallin family.

Glucosylation of benzyl alcohols by the cultured suspension cells of Nicotiana tabacum and Catharanthus roseus

The cultured cells of Nicotiana tabacum (white cells) converted regioselectively exogenous 2-, 3-, and 4-hydroxybenzyl alcohols into corresponding hydroxybenzyl-β-d-glucopyranoside. (RS)-1-Phenylethanol having chiral center in its substituent was also glucosylated to give 1-phenylethyl-β-d-glucopyranoside by the cultured cells of N. tabacum (white and green cells) and Catharanthus roseus. The glucosylation with the green cells of N. tabacum occurred enantioselectively to give the glucoside of (S)-alcohol preferentially, while the glucosylation with the white cells of N. tabacum and the C. roseus cells gave preferentially the glucoside of (R)-alcohol.

Asymmetric hydrolysis of enol esters with two esterases from Marchantia polymorpha

Abstract Two esterases participating in the asymmetric hydrolysis of α-alkylated enol acetates to α-chiral ketones were isolated from the cultured cells of Marchantia polymorpha . These two esterases had opposite enantioselectivities and both of them reversed the stereoselectivity of protonation into the enol intermediate in the hydrolysis when the chain length and the bulkiness of α-substituents were increased.

Stereochemistry in the reduction of the C=C bond of verbenone with a reductase from the cultured cells of Nicotiana tabacum

Reduction of the CC bond of verbenone by a reductase from the cultured cells of Nicotiana tabacuminvolves syn-addition of the hydrogen atom to the re-reface of the double bond; the hydrogen atoms participating in the reduction at C-2 and C-3 of verbenone originate from the pro-4Shydrogen of NADPH and the medium, respectively.

Biotransformation of hydroxycoumarins by the cultured cells of Nicotiana tabacum

Abstract Administration of hydroxycoumarins to the cultured cells of Nicotiana tabacum caused the ladder fragmentation of genomic DNA of the cells and the secretion of the intracellular phytoalexin, scopoletin, into the culture medium, and also the exogenous hydroxycoumarins were transformed into their corresponding β- d -glucosides. The glucosylation seems to be a defense reaction of the cells for detoxification of toxic hydroxycoumarins, because their glucosides did not cause the DNA fragmentation.