Ketone Reductase Activity Research Articles

Asymmetric Ketone Reduction Using Targeted Yeast Collections Isolated from Chinese Liquor Pits

Plenty of bacteria, yeasts, molds have been found in Chinese liquor pits. In order to investigate the applications of specifi c yeasts on ketone reduction, we screened some yeast strains in the Yeast Library of Chinese Liquor Pits in Chengdu Institute of Biology, Chinese Academy of Sciences, using f ive carbonyl compounds as potential substrates. The results indicated that the majority of the strains were capable of ketone reduction, and most of them obeyed the Prelog's rule. Some of the strains showed excellent stereo-selectivity, indicating high application potential in chiral drug intermediates production. For example, Williopsis sp. 2.045 transfered 3,5-bis(trif luoromethyl)-acetophenone into(S)-3,5-bis(trif luoromethyl)-1-phenethanol with over 99% yield and ee; Rhodotorula sp. 2.154 changed N-methyl-3-oxo-3-(thiophen-2-yl)propanamide into(S)-3-hydroxy-N-methyl-3-(thiophen-2-yl)propanamide with a yields of 95% and 99% ee. The results suggested that Chinese liquor pits contains lots of bioresources with high ketone reducing activity. It could be used as a targeted library in the screening of microbes with ketone reductase activity, which can highly improve the eff iciency of strain screening. Fig 6, Tab 6, Ref 24

Diplogelasinospora grovesii IMI 171018 immobilized in polyurethane foam. An efficient biocatalyst for stereoselective reduction of ketones

Diplogelasinospora grovesii has been reported as a very active biocatalyst in the reduction of ketones. Along the text, the properties of this filamentous fungus as an immobilized catalyst are described. For this purpose, several immobilization supports as agar and polyurethane foam were tested. Experimental assays were also performed to test different co-substrates for the regeneration of the required enzyme cofactor. The fungus immobilized in polyurethane foam lead to the most stable and active catalyst. This derivative, using i-PrOH as co-substrate, could be reused at least 18 times without appreciable activity loss (>90% activity remains). Kinetic runs experiments shown that the reduction of cyclohexanone, selected as model substrate, followed a pseudo-first kinetic order and that the rate controlling step was the mass transfer through the cell wall. The deactivation kinetic constants were also determined. The reduction of different chiral ketones showed that the ketone reductase activity followed the Prelog’s rule.

Monascus kaoliang CBS 302.78 immobilized in polyurethane foam using iso-propanol as co-substrate: Optimized immobilization conditions of a fungus as biocatalyst for the reduction of ketones

Monascus kaoliang was selected after a microbial screening as a highly active and selective whole cell catalyst for the reduction of ketones. In the present paper we describe the optimum growing conditions and an interesting immobilization procedure by adsorption in polyurethane foams (PUFs). This methodology is easy to perform and the immobilized catalyst is active, stable and reusable. The use of different co-substrates for cofactor regeneration was also tested and iso-propanol ( i-PrOH) was found as the best co-substrate, as it leads to a catalyst reusable for 17 cycles, displaying better NADH regeneration properties than others e.g., glucose (10 cycles) or saccharose (6 cycles). The reduction of different prochiral ketones showed that the ketone reductase activity of this mould follows the Prelog’s rule and kinetic experiments demonstrated that the process follows a pseudo-first kinetic order.

Irreversible blockade of sigma‐1 receptors by haloperidol and its metabolites in guinea pig brain and SH‐SY5Y human neuroblastoma cells

We evaluated the effect of haloperidol (HP) and its metabolites on [(3)H](+)-pentazocine binding to sigma(1) receptors in SH-SY5Y human neuroblastoma cells and guinea pig brain P(1), P(2) and P(3) subcellular fractions. Three days after a single i.p. injection in guinea pigs of HP (but not of other sigma(1) antagonists or (-)-sulpiride), [(3)H](+)-pentazocine binding to brain membranes was markedly decreased. Recovery of sigma(1) receptor density to steady state after HP-induced inactivation required more than 30 days. HP-metabolite II (reduced HP, 4-(4-chlorophenyl)-alpha-(4-fluorophenyl)-4-hydroxy-1-piperidinebutanol), but not HP-metabolite I (4-(4-chlorophenyl)-4-hydroxypiperidine), irreversibly blocked sigma(1) receptors in guinea pig brain homogenate and P(2) fraction in vitro. We found similar results in SH-SY5Y cells, which suggests that this process may also take place in humans. HP irreversibly inactivated sigma(1) receptors when it was incubated with brain homogenate and SH-SY5Y cells, but not when incubated with P(2) fraction membranes, which suggests that HP is metabolized to inactivate sigma(1) receptors. Menadione, an inhibitor of the ketone reductase activity that leads to the production of HP-metabolite II, completely prevented HP-induced inactivation of sigma(1) receptors in brain homogenates. These results suggest that HP may irreversibly inactivate sigma(1) receptors in guinea pig and human cells, probably after metabolism to reduced HP.

Studies on the Metabolic Fate of TA-510, a Hepatic Anti-inflammatory Agent. (III).Ildentitication of TA-510 Reductase in Human Liver.

To identify TA-510 reductase and obtain information on the stereochemical aspects, the reductase activity has been studied in subcellular fractions from 15 human livers, and finally purified using TA-510 enantiomers as substrates. 1. This activity was present in the cytosolic fraction and exhibited strong product stereoselectivity, i.e. both enantiomers were reduced exclusively to trans-alcohol (M1), but not cis-alcohol. Human liver microsomes showed practically no ketone reductase activity. 2. There was also a remarkable variation between the subjects in the substrate stereoselectivity, suggesting that racemic TA-510 was metabolized by at least two reductive enzymes with different stereochemical requirements. 3. The enzyme responsible for the reduction of (+)-TA-510 enantiomer was co-eluted with carbonyl reductase during purification steps, and separated from another enzyme with preference to (-)-TA-510 enantiomer as a substrate. Identity of (+) -TA-510 reductase and carbonyl reductase was also suggested by comparing the kinetic analysis and susceptibility to inhibitors of human liver cytosols and the purified enzyme. 4. (-)-TA-510 reductase was purified to a homogeneous protein and was shown as a monomeric protein with a molecular weight of 36 kDa. Amino acid sequences of five peptides obtained by proteolytic digestion of the purified enzyme were completely identical to the corresponding regions of previously reported 3α-hydroxysteroid/dihydrodiol dehydrogenase.

Purification and characterization of a novel cytosolic NADP(H)-dependent retinol oxidoreductase from rabbit liver

Rabbit liver cytosol exhibits very high retinol dehydrogenase activity. At least two retinol dehydrogenases were demonstrated to exist in rabbit liver cytosol, and the major one, a cytosolic NADP(H)-dependent retinol dehydrogenase (systematic name: retinol oxidoreductase) was purified about 1795-fold to electrophoretic and column chromatographic homogeneity by a procedure involving column chromatography on AF-Red Toyopearl twice and then hydroxyapatite. Its molecular mass was estimated to be 34 kDa by SDS-PAGE, and 144 kDa by HPLC gel filtration, suggesting that it is a homo-tetramer. The enzyme uses free retinol and retinal, and their complexes with CRBP as substrates in vitro. The optimum pH values for retinol oxidation of free retinol and CRBP-retinol were 8.8–9.2 and 8.0–9.0, respectively, and those for retinal reduction of free retinal and retinal-CRBP were the same, 7.0–7.6. K m for free retinol and V max for retinal formation were 2.8 μM and 2893 nmol/min per mg protein at 37°C (pH 9.0) and the corresponding values with retinol-CRBP as a substrate were 2.5 μM and 2428 nmol/min per mg protein at 37°C (pH 8.6); K m for free retinal and V max for retinol formation were 6.5 μM and 4108 nmol/min per mg protein, and the corresponding values with retinal-CRBP as a substrate were 5.1 μM and 3067 nmol/min per mg protein at 37°C, pH 7.4. NAD(H) was not effective as a cofactor. 4-Methylpyrazole was a weak inhibitor (IC 50=28 mM) of the enzyme, and ethanol was neither a substrate nor an inhibitor of the enzyme. This enzyme exhibits relatively broad aldehyde reductase activity and some ketone reductase activity, the activity for aromatic substitutive aldehydes being especially high and effective. Whereas, except in the case of retinol, oxidative activity toward the corresponding alcohols was not detected. This novel cytosolic enzyme may play an important role in vivo in maintaining the homeostasis of retinal, the substrate of retinoic acid synthesis, at least in rabbit liver, since a high concentration of retinol in liver and the lower K m of the enzyme for retinol force the oxidative reaction, while higher activity of retinal reductase at physiological pH forces the reductive reaction.

Ketone reductase activity and reduced haloperidol/haloperidol ratios in haloperidol-treated schizophrenic patients

Twenty schizophrenic patients were treated with a fixed haloperidol (HL) dose of 20 mg/day for 4 weeks. The conversion of HL to its reduced metabolite (reduced haloperidol, RH) occurs via the ketone reductase enzyme. RH is also converted back to HL by the cytochrome P450 2D6 isozyme. Ketone reductase activity can be measured in red blood cells. Plasma HL and RH levels were assayed by high performance liquid chromatography. Blood samples were obtained at baseline and during weeks 2 and 4 of HL therapy. Seventeen of 20 patients had ketone reductase values < 3. A significant correlation between ketone reductase activity and RH/HL plasma ratios was observed at week 4 in these 17 patients. Patients with ketone reductase activity |>3 could represent a subgroup of patients that metabolize HL differently. The wide interpatient variability observed with HL and RH plasma levels in HL-treated patients could reflect differences in ketone reductase activity and the metabolic status of debrisoquin hydroxylase (cytochrome P450IID6) in psychiatric patients.

Segmental differences in drug permeability, esterase activity and ketone reductase activity in the albino rabbit intestine.

Possible segmental differences in drug permeability as well as esterase and ketone reductase activities in the albino rabbit intestine were investigated. Beta adrenergic antagonists and timolol prodrugs spanning four orders of magnitude in distribution coefficient were used as model drugs. Drug penetration was evaluated in Ussing chambers using isolated segments of the duodenum, jejunum, ileum, ascending colon, descending colon, and rectum. Esterase and ketone reductase activities were determined in homogenates of the above segments using timolol ester prodrugs and levobunolol as substrates, respectively. The results indicate that the hydrophilic beta adrenergic antagonists atenolol and sotalol and moderately lipophilic metoprolol penetrated all intestinal segments equally well, whereas moderately lipophilic timolol and lipophilic propranolol, levobunolol and betaxolol were better absorbed from the large than from the small intestinal segments. Changes in lipophilicity exerted a more pronounced effect on the penetration of beta adrenergic antagonists in the large than the small intestinal segments. A similar pattern existed for timolol prodrugs. In addition to segmental differences in drug permeability, segmental differences in esterase and ketone reductase activities also existed. The level of esterase and ketone reductase activities in the small intestinal segments was, on average, 12 times and 5 times higher, respectively, than in the large intestinal segments. The implication of the above findings is that segmental differences in drug permeability and metabolism must be considered in the design of oral drug delivery systems.

Determination of haloperidol and reduced haloperidol in the plasma and blood of patients on depot haloperidol.

We developed a sensitive HPLC assay to measure haloperidol (HA) and its metabolite, reduced haloperidol (RH), in plasma and whole blood. The conditions under which HA might be converted to RH during collection and analysis of blood were examined. Provided the blood was kept at 0 degrees C, erythrocyte ketone reductase activity was insignificant. The solid phase extraction method did not generate RH. We studied ten patients taking 25-400 mg/month of HA decanoate and one patient for 4 weeks after the daily oral dose of 120 mg HA was ceased. In the patients on depot HA, the plasma and blood concentrations of HA were not significantly different (P greater than 0.1). For the first time, RH was detected in plasma patients on depot drug, but only in three cases. In contrast, RH was present in the blood of eight of these patients. The accumulation of RH in red blood cells was also evident in the patient on oral HA, in whom the mean ratio of RH concentrations in whole blood to plasma was 3.6 +/- 1.1. Plasma concentrations of HA correlated highly with total neuroleptic activity measured by a radioreceptor assay. Compared to plasma, analysis of concentrations of HA and RH in blood has the advantages of greater sensitivity, of using smaller volumes of blood and of avoiding the efflux of HA and RH during separation of plasma and red cells.

The in vitro ketone reduction of warfarin and analogues: Substrate stereoselectivity, product stereoselectivity and species differences

The in vitro metabolic ketone reduction of warfarin and its 4'-analogues acenocoumarol (4'-nitrowarfarin) and 4'-chlorowarfarin has been investigated using microsomal and cytosolic fractions of several species. Both subcellular fractions showed ketone reductase activity. The cytosolic fractions, in most species, exhibited strong substrate stereoselectivity as well as product stereoselectivity, i.e. the R(+)enantiomer was preferred as a substrate to be reduced mainly to the RS alcohol. Phenobarbital and methylcholanthrene induced cytosolic ketone reductase activity in the rat 5- to 11-fold and 3- to 7.4-fold, respectively. The microsomal fractions also showed substrate and product stereoselectivity. Contrary to the cytosolic fractions a general pattern for substrate and product stereoselectivity could not be seen. Stereoselectivity seemed species dependent (e.g. sheep vs bovine and pig). Rat liver microsomes showed practically no ketone reductase activity. Induction by phenobarbital or methylcholanthrene resulted in only a slight rise, if any, in rat microsomal ketone reductase activity. Both cytosolic and microsomal ketone reductases proved to be NADPH dependent. Substitution of the 4'-hydrogen of warfarin resulted in a change in reduction rates and in some cases, even in a change in substrate or product stereoselectivity. The data indicate that microsomal ketone reductases are different from cytosolic ketone reductases. Prelog's rule for product stereoselectivity of metabolic ketone reduction, when applied to the ketone reduction of the warfarin analogues did not agree with all data presented here.

A possible functional relationship between microsomal aromatic aldehyde-ketone reductase and hexose-6-phosphate dehydrogenase.

Aromatic ketone reductase activity of microsomes showed a unique cofactor requirement: Addition of NADP and glucose-6-phosphate was as effective as that of an artificial NADPH generating system, whereas NADPH alone served as a cofactor less efficiently. Microsomal aromatic ketone reductase, purified partially from guinea pig liver microsomes after solubilization with Triton X-100, reduced 5 beta-dihydrotestosterone, aromatic aldehydes, and ketones with NADPH as a cofactor. However, addition of hexose-6-phosphate dehydrogenase, purified from the same source, as an NADPH generator produced about 2 times higher activity than that of yeast glucose-6-phosphate dehydrogenase or NADPH alone.

Reduction of ketones in liver cytosol.

1. The predominant site for the hepatic reduction of aromatic and aliphatic ketones is the cytosol.2. Compounds in which at least one aromatic ring is adjacent to the carbonyl group are reduced to aryl-alkyl or aryl-aryl carbinols in NADPH-requiring reactions, whereas either NADH or NADPH serves as cofactor in the analogous reaction with aliphatic ketones.3. Acetophenone reductase activity is not enhanced in liver cytosol after treatment of rats with phenobarbital.4. Ketone reductase activity was found in the cytosol fractions of some non-hepatic tissues, particularly in that of kidney.