Secondary Alcohol Dehydrogenase Research Articles

A novel secondary alcohol dehydrogenase from Micrococcus luteus WIUJH20: purification, cloning, and properties

A novel NAD+‐dependent secondary alcohol dehydrogenase (2o‐ADH) has been purified to homogeneity from Micrococcus luteus WIUJH20 (M. luteus WIUJH20). It has a molecular weight of ~ 140 kDa as determined by gel filtration chromatography, and a molecular weight of ~ 34 kDa as determined by SDS‐PAGE. Thus, the enzyme is a homotetramer. The gene encodes for this enzyme has been amplified by PCR from the genome of M. luteus WIUJH20, cloned into pGEX2T expression vector, and recombinant protein overexpressed as a GST fusion protein (rGST‐2o‐ADH). Based on sequence homology, the enzyme is a NAD+‐dependent L‐3‐hydroxyacyl‐CoA dehydrogenase. But based on its catalytic activity, the enzyme belongs to secondary alcohol dehydrogenase. The rGST‐2o‐ADH fusion protein, the recombinant 2o‐ADH (r2o‐ADH) with GST‐tag removed, and the 2o‐ADH isolated from M. Luteus WIUJH20 had broad substrate specificity. Enzyme kinetics analysis showed that the r2o‐ADH possesses a Km and Vmax of 3.25 x 10−6 M and 150.3 μmol/min/mg respectively, when 12‐hydroxyoctadecanoic acid was the substrate. This r2o‐ADH prefers to use linear secondary alcohols as substrates over the cyclic secondary alcohols. The enzyme prefers long‐chain alcohol over short‐chain alcohol as shown by the decreasing in Km from 4‐heptanol (7 mM) to 6‐dodecanol (8 μM). Primary alcohols (particularly ethanol) are inhibitors to the 2o‐ADH and the inhibition constant Ki increases as the hydrocarbon chain length decreases.The research was supported in part by grants from USDA from USDA CSREES 04‐35504‐14712 and 02‐015470, Western Illinois University Foundation, College of Arts and Sciences and the Honors College at Western Illinois University.

Identification and Overexpression of a Bifunctional Aldehyde/Alcohol Dehydrogenase Responsible for Ethanol Production in Thermoanaerobacter mathranii

Thermoanaerobacter mathranii contains four genes, adhA, adhB, bdhA and adhE, predicted to code for alcohol dehydrogenases involved in ethanol metabolism. These alcohol dehydrogenases were characterized as NADP(H)-dependent primary alcohol dehydrogenase (AdhA), secondary alcohol dehydrogenase (AdhB), butanol dehydrogenase (BdhA) and NAD(H)-dependent bifunctional aldehyde/alcohol dehydrogenase (AdhE), respectively. Here we observed that AdhE is an important enzyme responsible for ethanol production in T. mathranii based on the constructed adh knockout strains. An adhE knockout strain fails to produce ethanol as a fermentation product, while other adh knockout strains showed no significant difference from the wild type. Further analysis revealed that the ΔadhE strain was defective in aldehyde dehydrogenase activity, but still maintained alcohol dehydrogenase activity. This showed that AdhE is the major aldehyde dehydrogenase in the cell and functions predominantly in the acetyl-CoA reduction to acetaldehyde in the ethanol formation pathway. Finally, AdhE was conditionally expressed from a xylose-induced promoter in a recombinant strain (BG1E1) with a concomitant deletion of a lactate dehydrogenase. Overexpressions of AdhE in strain BG1E1 with xylose as a substrate facilitate the production of ethanol at an increased yield.

Rapid molecular differentiation and genotypic heterogeneity among Candida parapsilosis and Candida orthopsilosis strains isolated from clinical specimens in Kuwait

Recent molecular studies have led to the recognition of three distinct species within the Candida parapsilosis complex, namely Candida parapsilosis, Candida orthopsilosis and Candida metapsilosis. As currently available yeast identification systems fail to differentiate these species, there is a paucity of information on their occurrence in different geographical regions. This study describes a simple PCR-based protocol for rapid discrimination among C. parapsilosis, C. orthopsilosis and C. metapsilosis strains by using primers derived from unique sequences within the internally transcribed spacer 1 (ITS1)-5.8 rRNA-ITS2 region. Retrospective analysis of 114 C. parapsilosis-complex isolates recovered from clinical specimens in Kuwait identified 109 as C. parapsilosis, five as C. orthopsilosis and none as C. metapsilosis. The results were further validated by PCR-RFLP patterns of the secondary alcohol dehydrogenase gene fragment. DNA sequencing of the ITS region and the D1/D2 regions of the 28S rRNA gene confirmed the species-specific identification of all five C. orthopsilosis strains. The amplicon length of the intergenic spacer between the 28S and 5S rRNA genes (IGS1) was also species-specific, and PCR-RFLP analyses of the IGS1 region identified two distinct genotypes among the five C. orthopsilosis strains, which corresponded with the ITS region sequence data. The three bloodstream C. orthopsilosis strains were confined to a single genotype. Among 81 randomly selected C. parapsilosis strains, two genotypes were detected by IGS1 region analyses, indicating limited genotypic heterogeneity among C. parapsilosis sensu stricto strains. As far as is known, this is the first report on the identification of C. orthopsilosis from a bloodstream infection in the Arabian Gulf region.

Cloning and characterization of 3‐hydroxyacyl‐CoA dehydrogenase from Micrococcus luteus WIUJH20

Soapstock is a by‐product of edible oil refining process with low cash value. It is rich in oleic and linoleic acids which can be converted to value‐added compounds by certain microorganisms. Nocardia cholesterolicum NRRL 5767 (N. cholesterolicum NRRL 5767) is a well‐known microbe that can transform oleic acid to 10‐hydroxyoctadecanoic acid (10‐HOOD, 85%) and 10‐oxo‐octadecanoic (10‐OXOD, 10%). The 10‐OXOD contaminant complicates downstream purification of 10‐OHOD. The conversion reactions are catalyzed by oleate dehydrogenase and secondary alcohol dehydrogenase. The long‐term goal is strain improvement of N. cholesterolicum NRRL 5767 by blocking 2‐ADH to create an industrially useful microbe for the production of 10‐OHOD from oleic acid. M. luteus was used for 2‐ADH purification due to its lack of oleate hydratase which hampered the purification of 2‐ADH from N. cholesterolicum NRRL 5767.The 2‐ADH from M. luteus WIUJH20 has been purified to homogeneity and its N‐terminal sequence determined. Using BLAST search tool, the 2‐ADH gene has been identified as belonging to the 3‐hydroxyacyl CoA dehydrogenase superfamily. The gene has been amplified by PCR and cloned into pGEX2T. The recombinant 2‐ADH protein has been overexpressed in E. coli, purified, and its Km and Vmax determined.Supported in part by grants from USDA CSREES 04‐35504‐14712 and 02‐015470.

Enantioselective synthesis of pure (R,R)-2,3-butanediol in Escherichia coli with stereospecific secondary alcohol dehydrogenases

We characterized the activity and stereospecificity of four secondary alcohol dehydrogenases (sADHs) towards acetoin reduction and constructed synthetic pathways in E. coli to produce enantiomerically pure (R,R)-2,3-butanediol (2,3-BDO) from glucose with a titer of 6.1 g/L (enantio purity >99%), and yield of 0.31 g product/g glucose (62% of theoretical maximum).

Purification and characterization of cytoplasmic NAD+-dependent polypropylene glycol dehydrogenase fromStenotrophomonas maltophilia

The oxidizing enzyme NAD(+)-dependent polypropylene glycol dehydrogenase (PPG-DH) was purified to homogeneity from the cytoplasmic fraction of Stenotrophomonas maltophilia grown on polypropylene glycol (diol type) 2000. The purified enzyme consisted of a homotetrameric protein (37 kDa subunit) with a molecular mass of around 154 kDa. The N-terminal amino acid sequence (25 residues) showed similarity to the sequences of NAD(+)-dependent secondary alcohol dehydrogenases and NADH-dependent reductases. The enzyme preferentially oxidized medium-chain secondary alcohols, di- and tri-propylene glycols and polypropylene glycols including those with secondary alcohol groups in their molecular structure. Consequently, the enzyme was classified into a group of NAD(+)-dependent secondary alcohol dehydrogenases.

Crystal Structures of F420-dependent Glucose-6-phosphate Dehydrogenase FGD1 Involved in the Activation of the Anti-tuberculosis Drug Candidate PA-824 Reveal the Basis of Coenzyme and Substrate Binding

The modified flavin coenzyme F(420) is found in a restricted number of microorganisms. It is widely distributed in mycobacteria, however, where it is important in energy metabolism, and in Mycobacterium tuberculosis (Mtb) is implicated in redox processes related to non-replicating persistence. In Mtb, the F(420)-dependent glucose-6-phosphate dehydrogenase FGD1 provides reduced F(420) for the in vivo activation of the nitroimidazopyran prodrug PA-824, currently being developed for anti-tuberculosis therapy against both replicating and persistent bacteria. The structure of M. tuberculosis FGD1 has been determined by x-ray crystallography both in its apo state and in complex with F(420) and citrate at resolutions of 1.90 and 1.95 A(,) respectively. The structure reveals a highly specific F(420) binding mode, which is shared with several other F(420)-dependent enzymes. Citrate occupies the substrate binding pocket adjacent to F(420) and is shown to be a competitive inhibitor (IC(50) 43 microm). Modeling of the binding of the glucose 6-phosphate (G6P) substrate identifies a positively charged phosphate binding pocket and shows that G6P, like citrate, packs against the isoalloxazine moiety of F(420) and helps promote a butterfly bend conformation that facilitates F(420) reduction and catalysis.

Non-equivalence of Hydrogen Transfer from Glucose to the pro-R and pro-S Methylene Positions of Ethanol during Fermentation by Leuconostoc mesenteroides Quantified by 2H NMR at Natural Abundance

The anaerobic fermentation of glucose by Leuconostoc mesenteroides via the reductive pentose phosphate pathway leads to the accumulation of lactic acid and ethanol. The isotope redistribution coefficients (a(ij)) that characterize the specific derivation of each hydrogen atom in ethanol in relation to the non-exchangeable hydrogen atoms in glucose and the medium water have been determined using quantitative (2)H NMR. First, it is confirmed that the hydrogens of the methylene group are related only to the 1 and 3 positions of glucose via the NAD(P)H pool and not to the 4 position, in contrast to ethanol produced by Saccharomyces cerevisiae. Second, it is found that the conversion factors (C(f)) for the transfer of hydrogen to the pro-S and pro-R positions of the methylene group are not equivalent: the C(f)-1-R:C(f)-1-S ratio is 2.1, whereas the C(f)-3-R:C(f)-3-S ratio is 0.8. It is shown that this non-equivalence is not determined by the stereochemistry of the terminal NADH- and NADPH-dependent alcohol dehydrogenases, but is dependent on the cofactor selectivities of the reductive and oxidative steps of the reduced nucleotide cycle.

Secondary Alcohol Dehydrogenase in Micrococcus luteus (WIUJH‐20)

In our efforts to screen microorganisms that can convert agricultural commodities to value‐added products, we isolated a strain of Micrococcus luteus (WIUJH‐20) that effectively converted 10‐ or 12‐ hydroxysteric acid to their corresponding keto‐derivatives. The results suggested that this strain is an excellent source for isolation of a secondary alcohol dehydrogenase. The secondary alcohol dehydrogenase has been purified to homogeneity by several chromatographic steps. The native secondary alcohol dehydrogenase appears to be a homotetramer with a molecular weight of about 160 kDa. The isoelectric point of the enzyme is about 4.8 as determined by two‐dimensional gel electrophoresis. N‐terminal amino acid sequencing revealed that it is quite different from that of cytosolic secondary alcohol dehydrogenases. Very likely, the secondary alcohol dehydrogenase we are dealing with belongs to the membrane‐bound secondary alcohol dehydrogenase for the fact that both the substrate (10‐hydro‐ or 12‐hydroxysteric acids) and the product (10‐oxo‐ or 12‐oxo derivatives) are solid at room temperature preventing translocation across the membrane. We are doing peptide mapping and N‐terminal amino acid sequencing of the internal peptide fragments to design primers to amplify the gene by PCR for further study. This research is supported by the Western Illinois University Centennial Honors College.

Versatile bioelectronic interfaces on flexible non-conductive substrates

Bioelectronic interfaces that establish electrical communication between redox enzymes and electrodes have potential applications as biosensors, biocatalytic reactors, and biological fuel cells. These interfaces are commonly formed on gold films deposited using physical vapor deposition (PVD) or chemical vapor deposition (CVD). PVD and CVD require deposition of a primer layer, such as titanium or chromium, and require the use of expensive equipment and cannot be used on a wide range of substrates. This paper describes a versatile new bench-top method to form bioelectronic interfaces containing a gold film, electron mediator, cofactor, and dehydrogenase enzyme (secondary alcohol dehydrogenase, and sorbitol dehydrogenase) on nonconductive substrates such as polystyrene and glass. The method combines layer-by-layer deposition of polyelectrolytes, electroless metal deposition, and directed molecular self-assembly. Cyclic voltammetry, chronoamperometry, field emission X-ray dispersive spectroscopy, scanning electron microscopy, and atomic force microscopy were used to characterize the bioelectronic interfaces. Interfaces formed on flexible polystyrene slides were shown to retain their activity after bending to a radius of curvature of 18mm, confirming that the approach can be applied on cheap and flexible substrates for applications where traditional wafer-scale electronics is not suitable, such as personal or structural health monitors and rolled microtube biosensors.

Activity and selectivity of W110A secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus in organic solvents and ionic liquids: mono- and biphasic media

The asymmetric reduction of hydrophobic phenyl-ring-containing ketones and the enantiospecific kinetic resolution of the corresponding racemic alcohols catalyzed by Thermoanaerobacter ethanolicus W110A secondary alcohol dehydrogenase were performed in mono- and biphasic systems containing either organic solvents or ionic liquids. Both yield and enantioselectivity for these transformations can be controlled by changing the reaction medium. The enzyme showed high tolerance to both water-miscible and -immiscible solvents, which allows biotransformations to be conducted at high substrate concentrations.

Engineered Synthetic Pathway for Isopropanol Production in Escherichia coli

A synthetic pathway was engineered in Escherichia coli to produce isopropanol by expressing various combinations of genes from Clostridium acetobutylicum ATCC 824, E. coli K-12 MG1655, Clostridium beijerinckii NRRL B593, and Thermoanaerobacter brockii HTD4. The strain with the combination of C. acetobutylicum thl (acetyl-coenzyme A [CoA] acetyltransferase), E. coli atoAD (acetoacetyl-CoA transferase), C. acetobutylicum adc (acetoacetate decarboxylase), and C. beijerinckii adh (secondary alcohol dehydrogenase) achieved the highest titer. This strain produced 81.6 mM isopropanol in shake flasks with a yield of 43.5% (mol/mol) in the production phase. To our knowledge, this work is the first to produce isopropanol in E. coli, and the titer exceeded that from the native producers.

Production of 14‐Oxo‐cis‐11‐eicosenoic Acid from Lesquerolic Acid by Sphingobacterium multivorum NRRL B‐23212

AbstractThe objective of this study was to explore the extent of microbial conversion of lesquerolic acid (14‐hydroxy‐cis‐11‐eicosenoic acid; LQA) by whole cell catalysis and to identify the newly converted products. Among compost isolates including NRRL strains B‐23212 (Sphingobacterium multivorum), B‐23213 (Acinetobacter sp.), B‐23257 (Enterobacter cloacae B), B‐23259 (Escherichia sp.) and B‐23260 (Pseudomonas aeruginosa) the S.multivorum strain was the only microorganism that converted LQA to produce a new product identified as 14‐oxo‐cis‐11‐eicosenoic acid by GC‐MS and NMR analyses. The conversion yield was 47.4% in 48 h at 200 rpm and 28°C in small shake flask experiments. In comparison, both Acinetobacter and Pseudomonas strains failed to convert LQA to major new products but used LQA apparently as an energy source during fermentation. For structural analysis, 6.88 g of 14‐oxo‐cis‐11‐eicosenoic acid was produced from converting 11 g LQA (a 62% yield) in 72 h at 200 rpm and 28 °C in Fernbach flasks using 18‐h‐old NRRL B‐23212 cultures and an improved medium that also contained EDTA and glycerol in lieu of glucose as carbon source. NRRL B‐23212 was further identified by 16S rRNA gene sequence analysis as a unique strain of S. multivorum. Therefore, S. multivorum NRRL B‐23212 possesses an enzymatic activity presumably a secondary alcohol dehydrogenase for converting LQA to produce 14‐oxo‐cis‐11‐eicosenoic acid, a first report that demonstrates the functional modification of LQA by whole cell catalysis.

Renewable Dehydrogenase-Based Interfaces for Bioelectronic Applications

Bioelectronic interfaces that establish electrical communication between redox enzymes and electrodes have potential applications as biosensors, biocatalytic reactors, and biological fuel cells. However, these interfaces contain labile components, including enzymes and cofactors, which have limited lifetimes and must be replaced periodically to allow long-term operation. Current methods to fabricate bioelectronic interfaces do not allow facile replacement of these components, thus limiting the useful lifetime of the interfaces. In this paper we describe a versatile new fabrication approach that binds the enzymes and cofactors using reversible ionic interactions. This approach allows the interface to be removed via a simple pH change and then replaced to fully regenerate the biocatalytic activity. The positively charged polyelectrolyte poly(ethylenimine) was used to ionically bond a dehydrogenase enzyme and its cofactor to a gold electrode that was functionalized with 3-mercaptopropionic acid and the electron mediator toluidine blue O. By reducing the pH, the surface-bound 3-mercaptopropionic acid was protonated, disrupting the ionic bonds and releasing the enzyme-modified polyelectrolyte. After neutralization, fresh enzyme and cofactor were bound, regenerating the bioelectronic interface. Cyclic voltammetry, chronoamperometry, constant potential amperometry, electrochemical impedance spectroscopy, and Fourier transform infrared spectroscopy analyses were used to characterize the bioelectronic interfaces. For the two enzymes tested (secondary alcohol dehydrogenase and sorbitol dehydrogenase) and their respective cofactors (beta-nicotinamide adenine dinucleotide phosphate and beta-nicotinamide adenine dinucleotide), the reconstituted interface exhibited a surface coverage, an electron-transfer coefficient, and a turnover rate similar to those of the original interface.

Mutation of Tyr-218 to Phe in Thermoanaerobacter ethanolicus Secondary Alcohol Dehydrogenase: Effects on Bioelectronic Interface Performance

Bioelectronic interfaces that facilitate electron transfer between the electrode and a dehydrogenase enzyme have potential applications in biosensors, biocatalytic reactors, and biological fuel cells. The secondary alcohol dehydrogenase (2 degrees ADH) from Thermoanaerobacter ethanolicus is especially well suited for the development of such bioelectronic interfaces because of its thermostability and facile production and purification. However, the natural cofactor for the enzyme, beta-nicotinamide adenine dinucleotide phosphate (NADP+), is more expensive and less stable than beta-nicotinamide adenine dinucleotide (NAD+). PCR-based, site-directed mutagenesis was performed on 2 degrees ADH in an attempt to adjust the cofactor specificity toward NAD+ by mutating Tyr218 to Phe (Y218F 2 degrees ADH). This mutation increased the Km(app) for NADP+ 200-fold while decreasing the Km(app) for NAD+ 2.5-fold. The mutant enzyme was incorporated into a bioelectronic interface that established electrical communication between the enzyme, the NAD+, the electron mediator toluidine blue O (TBO), and a gold electrode. Cyclic voltammetry, impedance spectroscopy, gas chromatography, mass spectrometry, constant potential amperometry, and chronoamperometry were used to characterize the mutant and wild-type enzyme incorporated in the bioelectronic interface. The Y218F 2 degrees ADH exhibited a fourfold increase in the turnover ratio compared to the wild type in the presence of NAD+. The electrochemical and kinetic measurements support the prediction that the Rossmann fold of the enzyme binds to the phosphate moiety of the cofactor. During the 45 min of continuous operation, NAD+ was electrically recycled 6.7 x 10(4) times, suggesting that the Y218F 2 degrees ADH-modified bioelectronic interface is stable.

Xerogel‐Encapsulated W110A Secondary Alcohol Dehydrogenase from Thermoanaerobacter ethanolicus Performs Asymmetric Reduction of Hydrophobic Ketones in Organic Solvents

To gel well: The asymmetric reduction of hydrophobic ketones by xerogel-immobilized W110A secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus (TeSADH) in organic solvents affords their S-configured alcohols in yields comparable with those achieved by using the free enzyme, and, in some cases, with higher enantioselectivities. R=phenyl-ring-containing substituent.

Genotyping of Candida orthopsilosis Clinical Isolates by Amplification Fragment Length Polymorphism Reveals Genetic Diversity among Independent Isolates and Strain Maintenance within Patients

Candida parapsilosis former groups II and III have recently been established as independent species named C. orthopsilosis and C. metapsilosis, respectively. In this report, 400 isolates (290 patients) previously classified as C. parapsilosis by conventional laboratory tests were screened by BanI digestion profile analysis of the secondary alcohol dehydrogenase gene fragment and by amplification fragment length polymorphism (AFLP). Thirty-three strains collected from 13 patients were identified as C. orthopsilosis, thus giving the first retrospective evidence that C. orthopsilosis was responsible for 4.5% of the infections/colonization attributed to C. parapsilosis. AFLP was proven to unambiguously identify C. orthopsilosis at the species level and efficiently delineate intraspecific genetic relatedness. A high percentage of polymorphic AFLP bands was observed for independent isolates collected from each patient. Statistical analysis of the pairwise genetic distances and bootstrapping revealed that clonal reproduction and recombination both contribute to C. orthopsilosis genetic population structure. AFLP patterns of sequential isolates obtained from two patients demonstrated that a successful strain colonization within the same patient occurred, as revealed by strain maintenance in various body sites. No association between AFLP markers and drug resistance was observed, and none of the clinical C. orthopsilosis isolates were found to produce biofilm in vitro.

A Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase mutant derivative highly active and stereoselective on phenylacetone and benzylacetone

The secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus 39E (TeSADH) is highly thermostable and solvent-stable, and it is active on a broad range of substrates. These properties make TeSADH an excellent template to engineer an industrial catalyst for chiral chemical synthesis. (S)-1-Phenyl-2-propanol was our target product because it is a precursor to major pharmaceuticals containing secondary alcohol groups. TeSADH has no detectable activity on this alcohol, but it is highly active on 2-butanol. The structural model we used to plan our mutagenesis strategy was based on the substrate's orientation in a horse liver alcohol dehydrogenase*p-bromobenzyl alcohol*NAD(+) ternary complex (PDB entry 1HLD). The W110A TeSADH mutant now uses (S)-1-phenyl-2-propanol, (S)-4-phenyl-2-butanol and the corresponding ketones as substrates. W110A TeSADH's kinetic parameters on these substrates are in the same range as those of TeSADH on 2-butanol, making W110A TeSADH an excellent catalyst. In particular, W110A TeSADH is twice as efficient on benzylacetone as TeSADH is on 2-butanol, and it produces (S)-4-phenyl-2-butanol from benzylacetone with an enantiomeric excess above 99%. W110A TeSADH is optimally active at 87.5 degrees C and remains highly thermostable. W110A TeSADH is active on aryl derivatives of phenylacetone and benzylacetone, making this enzyme a potentially useful catalyst for the chiral synthesis of aryl derivatives of alcohols. As a control in our engineering approach, we used the TbSADH*(S)-2-butanol binary complex (PDB entry 1BXZ) as the template to model a mutation that would make TeSADH active on (S)-1-phenyl-2-propanol. Mutant Y267G TeSADH did not have the substrate specificity predicted in this modeling study. Our results suggest that (S)-2-butanol's orientation in the TbSADH*(S)-2-butanol binary complex does not reflect its orientation in the ternary enzyme-substrate-cofactor complex.

Asymmetric Reduction and Oxidation of Aromatic Ketones and Alcohols Using W110A Secondary Alcohol Dehydrogenase from Thermoanaerobacter ethanolicus

An enantioselective asymmetric reduction of phenyl ring-containing prochiral ketones to yield the corresponding optically active secondary alcohols was achieved with W110A secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus (W110A TESADH) in Tris buffer using 2-propanol (30%, v/v) as cosolvent and cosubstrate. This concentration of 2-propanol was crucial not only to enhance the solubility of hydrophobic phenyl ring-containing substrates in the aqueous reaction medium, but also to shift the equilibrium in the reduction direction. The resulting alcohols have S-configuration, in agreement with Prelog's rule, in which the nicotinamide-adenine dinucleotide phosphate (NADPH) cofactor transfers its pro-R hydride to the re face of the ketone. A series of phenyl ring-containing ketones, such as 4-phenyl-2-butanone (1a) and 1-phenyl-1,3-butadione (2a), were reduced with good to excellent yields and high enantioselectivities. On the other hand, 1-phenyl-2-propanone (7a) was reduced with lower ee than 2-butanone derivatives. (R)-Alcohols, the anti-Prelog products, were obtained by enantiospecific oxidation of (S)-alcohols through oxidative kinetic resolution of the rac-alcohols using W110A TESADH in Tris buffer/acetone (90:10, v/v).

New Insights into Acetone Metabolism ▿

New Insights into Acetone Metabolism ^▿