Kazutoshi Ushio

Stereochemical control in yeast reduction

Abstract A substrate which is subjected to yeast reduction has been modified by iodination or phenylsulfonylation. Both substituents improves chemical and optical yields and control the stereochemical course of the reduction.

Stereochemical control in microbial reduction 4. Effect of cultivation conditions on the reduction of β-keto esters by methylotrophic yeasts.

Abstract When β-keto esters are reduced by methanol grown yeast, drastic shift of the enantiomer excess of the products toward D-isomer formation is caused compared with those by glucose grown cells.

Asymmetric reduction of ketones by glycerol dehydrogenase from geotricum

Abstract Glycerol dehydrogenase from Geotricum was used as a catalyst for asymmetric reduction of ketones. A 2-propanol-NAD + couple was employed to supply NADH.

Coenzymic function of 1- or N6-substituted analogs of adenosylcobalamin in the diol dehydratase reaction

Five analogs of adenosylcobalamin modified in the adenine moiety of the Coβ ligand were synthesized, and their coenzymic properties were studied with diol dehydratase of Klebsiella pneumoniae ATCC 8724. N6-Methyladenosylcobalamin showed a coenzymic activity of 17% that of adenosylcobalamin and a Km value of 4.2 μM. Like the natural holoenzyme, the complex between apoenzyme and this analog acted on glycerol and underwent suicide inactivation, although the rate of inactivation was much slower than that with the natural holoenzyme. Almost no spectral change was observed in the visible region during the course of the inactivation by glycerol, suggesting the formation of some new alkylcobalamin-like species as observed in the inactivation of the natural holoenzyme by glycerol. 1-Methyladenosylcobalamin and N6-ethyladenosylcobalamin did not show detectable coenzymic activity in either 1,2-propanediol- or glycerol-dehydration reaction, although their affinity for the enzyme (Ki = 5.3 and 5.4 μM, respectively) was not so much different from that for N6-methyladenosyleobalamin. 1,N6-Ethenoadenosylcobalamin was also inactive and showed very low affinity for the enzyme (Ki ≈ 35 μM), indicating that simultaneous blockage of both 1- and N6-positions inhibits the interaction with the enzyme. 3,N4-Ethenocytidylcobalamin was inactive, although it binds to the enzyme as tightly (Ki = 1.4 μM) as does adenosylcobalamin.

Formation of thieno[3,2-g]pterines from the molybdenum cofactor

A fluorescent oxidation product of the molybdenum cofactor was isolated from Escherichia coli nitrate reductase (EC 1.9.6.1) and bovine milk xanthine oxidase (EC 1.2.3.2), which showed a visible absorption band at 395 nm and was dephosphorylated by alkaline phosphatase but not by phosphodiesterase I. The dephosphorylated species was oxidized by periodate to thieno[3,2-g]pterin-2-carbaldehyde which was quantitatively converted to thieno[3,2-g]pterin-2-carboxylic acid by subsequent treatment with Ag2O in 2 N NaOH. These results indicate that the oxidation product of the molybdenum cofactor is a thieno[3,2-g]pterin derivative with an unidentified side chain in the 2 position.

An electron paramagnetic resonance study on the mechanism-based inactivation of adenosylcobalamin-dependent diol dehydrase by glycerol and other substrates

Adenosylcobalamin-dependent diol dehydrase undergoes mechanism-based inactivation by glycerol or other substrates during catalysis. X-band electron paramagnetic resonance spectra of holoenzyme were measured at -130 degrees C after reaction with such substrates. After short time of incubation, broad signals assigned to low-spin Co(II) of cob(II)alamin and doublet signals assigned to an organic radical intermediate derived from each substrate were observed with 1,2-propanediol, 1,2-ethanediol, glycerol and meso-2,3-butanediol with the magnitude of their exchange interaction (J-value) decreasing in this order. A substrate with the smaller magnitude of exchange interaction between low-spin Co(II) and an organic radical intermediate seems to be an efficient mechanism-based inactivator. Since the magnitude of exchange interaction decreases with the distance between radical species in a radical pair, these results suggest that a stabilizing effect of holoenzyme on radical intermediates during reactions decreases with the distance between Co(II) and a radical.

Identification of a dephosphorylated oxidation product of the molybdenum cofactor as 2-(1,2-dihydroxyethyl)thieno[3,2-g]pterin

A new method was developed for the synthesis of 2-(1,2-dihydroxyethyl)thieno[3,2-g]pterin and related 2-substituted thienopterins. A dephosphorylated fluorescent oxidation product of the molybdenum cofactor isolated from xanthine oxidase (EC 1.2.3.2) was identified as 2-(1,2-dihydroxyethyl)thieno[3,2-g]pterin by comparison of electronic and fluorescence spectra and TLC behaviors with those of the synthetic compound.