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Thus far, only a handful of bacterial strains that can independently degrade and utilize benzo[a]pyrene (BaP) as the sole carbon source have been isolated and characterized. Here, three new bacterial strains, JBZ1A, JBZ2B, and JBZ5E, were isolated from contaminated soil and, using 16S rRNA sequencing, were identified as Bradyrhizobium japonicum, Micrococcus luteus, and Bacillus cereus, respectively. The growth ability of each individual strain and a consortium of all strains in the presence of BaP (4–400 µmol·l−1, pH 7, 37°C) was identified by the doubling time (dt). The results illustrated that dt decreased with increasing BaP concentrations for individual strain and the consortium. The optimum growth conditions of the consortium were 37°C, 0.5% NaCl (w/v), and pH 7. Under these conditions, the degradation rate was 1.06 µmol·l−1·day−1, whereas that of individual strains ranged 0.9–0.38 µmol·l−1·day−1. B. cereus had the strongest contribution to the consortium’s activity, with a degradation rate of 0.9 µmol·l−1·day−1. The consortium could also remove BaP spiked with soil, but at a lower rate (0.01 µmol.l−1.day−1). High-performance liquid chromatography–high-resolution tandem mass spectrometry permitted the detection of the metabolites of these strains, and a biodegradation pathway has been proposed.

We developed a concurrent cyclic deracemisation approach for secondary alcohols that combines a non-stereospecific oxidation step and a stereoselective reduction step using two mutants of Thermoanaerobacter pseudoethanolicus secondary alcohol dehydrogenase (TeSADH) that exhibit various extents of stereoselectivities. In this approach, W110G TeSADH, a sparingly stereoselective mutant, performs the non-stereospecific oxidation step and W110V/G198D TeSADH performs the stereoselective reduction step. The use of orthogonal cofactor regeneration systems allowed for the spontaneous operation of these mutants. (S)-Configured alcohols were obtained in moderate ee's from their racemates using this strategy. To our knowledge, this report provides the first example of a fully enzymatic cyclic deracemisation with a stereoselective reduction step (CD-RS) for alcohols. This approach was further improved into a deracemisation strategy via stereoinversion using concurrent (R)-selective I86A TeSADH-catalysed oxidation that leaves (S)-alcohols untouched and W110V/G198D TeSADH-catalysed stereoselective reduction of the resultant ketone intermediates into the corresponding (S)-configured alcohols. The latter strategy enabled quantitative production of (S)-1-phenylethanol in >99% ee from its racemate. Overall, we show the superiority of the stereoinversion deracemisation approach for alcohols when compared with cyclic deracemisation, which is mainly due to the elimination of futile cycles in the former.

To date, only a handful of bacterial strains that can independently degrade and utilize benzo[a]pyrene (BaP) as the sole carbon source has been isolated and characterized. Here, three new bacterial strains—JBZ1A, JBZ2B, and JBZ5E—were isolated from contaminated soil and, using 16S rRNA sequencing, were identified as Brad rhizobium japonicum, Micrococcus luteus, and Bacillus cereus, respectively. The growth ability of each individual strain and a consortium of all strains in the presence of BaP (4–400 µmol·L−1, pH 7, 37 °C) was identified by the doubling time (dt). The results illustrate that dt decreased with increasing BaP concentrations for individual strains and the consortium. The optimum growth conditions of the consortium were 37 °C, 0.5% NaCl (w/v), and pH 7. Under these conditions, the degradation rate was 1.06 µmol·L−1·day−1, whereas that of individual strains ranged from 0.9 to 0.38 µmol·L−1·day−1. B. cereus had the strongest contribution to the consortium’s activity, with a degradation rate of 0.9 µmol·L−1·day−1. The consortium could also remove BaP spiked with soil but at a lower rate (0.01 µmol L−1.day−1). High-performance liquid chromatography–high-resolution tandem mass spectrometry permitted the detection of the metabolites of these strains, and a biodegradation pathway is proposed.

Polycyclic aromatic hydrocarbons (PAHs), which consist of low-molecular-weight PAHs (LMW-PAHs) and high-molecular-weight PAHs (HMW-PAHs), form an important class of pollutants. Pyrene and benzo[a]pyrene (BaP) are the main pollutants belonging to HMW-PAHs, and their degradation by microorganisms remains an important strategy for their removal from the environments. Extensive studies have been carried out on the isolation and characterisation of microorganisms that actively degrade LMW-PAHs, and to a certain extent, the HMW-PAH pyrene. However, so far, limited work has been carried out on BaP biodegradation. BaP consists of five fused aromatic rings, which confers this compound a high stability, rendering it less amenable to biodegradation. The current review summarizes the emerging reports on BaP biodegradation. More specifically, work carried out on BaP bacterial degradation and current knowledge gaps that limit our understanding of BaP degradation are highlighted. Moreover, new avenues of research on BaP degradation are proposed, specifically in the context of the development of “omics” approaches

Alcohol dehydrogenases (ADHs) represent an important type of enzymes that have significant applications as biocatalysts. Secondary ADHs from Thermoanaerobacter pseudoethanolicus ( Te SADH) and from Thermoanaerobacter brockii ( Tb SADH) are well-known as robust catalysts. However, and like most other ADHs, these enzymes suffer from their high substrate specificities (i.e., limited substrate scope), which to some extent restricts their use as biocatalysts. This review discusses recent efforts to expand the substrate scope and tune enantioselectivity of Te SADH and Tb SADH using site-directed mutagenesis and directed evolution. Various examples of asymmetric synthesis of optically active alcohols using both enzymes are highlighted. Moreover, the unique thermal stability and organic solvent tolerance of these enzymes is illustrated by their concurrent inclusion with other interesting reactions to synthesize optically active alcohols and amines. For instance, Te SADH has been used in quantitative non-stereoselective oxidation of alcohols to accomplish deracemization of alcohols via cyclic deracemization and in racemization of enantiopure alcohols to accomplish a bienzymatic dynamic kinetic resolution.

Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants from incomplete combustion and petroleum products. As the molecular weight increases, PAHs become more recalcitrant to biodegradation. A bacterial strain capable of metabolizing the four fused aromatic ring PAH pyrene was isolated and characterized. The analysis of 16S rRNA gene revealed that it belongs to Achromobacter xylosoxidans species. A. xylosoxidans PY4 can utilize pyrene as the sole source of carbon. PY4 has a doubling time (dt) of less than 1 day when it grows in the presence of 1–5 mg l−1 pyrene, a dt range similar to that of the most efficient pyrene biodegrading bacteria described so far. The optimal pyrene degradation conditions are at pH 7–9, 37–40 °C, and 0–2.5% NaCl. PY4 also utilizes salicylic acid, catechol, naphthalene, anthracene and phenanthrene. PY4 degrades more than 50% of 100 mg l−1 of pyrene, within the first 15 days, at a rate of 0.069 day−1, R2 = 0.99. The metabolites include monohydroxypyrene, 1-methoxyl-2-H-benzo[h]chromene-2-carboxylic acid, 9,10-phenanthrenequinone, 1-methoxyl-trans-2′-carboxybenzalpyruvate, and dibutyl-phthalate. Up-expressed proteins in response to pyrene are involved in cell homeostasis, genetic information synthesis and storage, and chemical stress. Among these proteins are 4-hydroxyphenylpyruvate dioxygenase and homogentisate 1,2-dioxygenase, involved in the lower pyrene degradation pathway.

The authors acknowledge the support provided by the Deanship of Scientific Research (DSR) at King Fahd University of Petroleum and Minerals (KFUPM) for funding this work under project number IN151032. They also acknowledge the supported by baseline research fund to S.M.H. by King Abdullah University of Science and Technology.

A trial was conducted to check effect of adding gum Arabic (GA) instead of egg yolk (EY) as a cryoprotectant for stallion sperm. Two experiments were designed; experiment I tested adding 3 levels of nonheated GA (i.e., 3, 6 and 9 g/100 mL diluents) in HF-20 extender. However, in experiment II the same levels were tested except that GA was heated at 80 °C for 60 min. HF-20 containing 10% of EY was used as control. In experiment I, sperm frozen in HF-20 containing nonheated GA exhibited lower percentages of motile sperm, progressively motile sperm and sperm with intact plasma membranes, vitality rate, and acrosome integrity after cooling or after deep freezing. Frozen semen in HF-20 containing 3–6% of preheated GA in experiment II maintained sperm motility at 46–50% and elevated progressive motility at 27%. The semen diluted in preheated GA (6%) and frozen exhibited a fertility rate of 40% (2/5). A similar fertility rate (40%) was found in the control semen (i.e. 10%) compared to those that were inseminated with frozen semen in preheated 3% GA (20%, 1/5). These results suggest that preheated GA could be used as an alternative cryoprotectant for cryopreserving stallion sperm.

The interaction of gold(III) complexes [Au(en)Cl2]Cl, [Au(en)2]Cl3, [Au(cis-DACH)Cl2]Cl, and [Au(cis-DACH)2]Cl3 (en = ethylenediamine, DACH = cis-1,2-diaminocyclohexane) with biologically important thiols, such as glutathione (GSH), dl-penicillamine (PSH), mercaptoacetic acid (MAA), and N-(2-mercaptopropionyl)glycine (MPG), has been studied using 1H, 13C NMR, UV–vis spectroscopy and electrochemistry in aqueous solution. Kinetic data revealed that the reactivity of their substitution reaction followed the order: [Au(en)Cl2]+ > [Au(en)2]3+ > [Au(cis-DACH)Cl2]+ > [Au(cis-DACH)2]3+. The thiol reactivity increased with decreasing its size, viz. MAA ≫ MPG > PSH > GSH. Square wave stripping voltammetry displayed peaks for Au(III) and Au(I) at +0.875 V and +1.4 V respectively. The interaction of the complexes with thiols resulted in reduction of gold(III) to gold(I) and thiol ligands (RSH) were oxidized to disulfide (RSSR).

Here, ketones bearing two bulky substituents, named bulky-bulky ketones, were successfully reduced to their corresponding optically-enriched alcohols using various mutants of Thermoanaerobacter pseudoethanolicus secondary alcohol dehydrogenase (TeSADH). Substituted 2-tetralones in particular were reduced to 2-tetralols with high conversion and high enantioselectivity. The pharmacological importance of substituted 2-tetralols as key drug-building blocks makes our biocatalytic reduction method a highly essential tool. We showed that changing the position of the substituent on the aromatic ring of 2-tetralones impacted their binding affinity and the reaction maximum catalytic rate. Docking studies with several TeSADH mutants explained how the position of the substituent on the tetralone influences the binding orientation of substituted 2-tetralones and their reaction stereoselectivity.

Optical rotatory dispersion (ORD) spectra are reported for isolated and solvated (R)-3-methylcyclohexanone (R-3MCH) in 10 solvents, of wide polarity range, and over the spectral range 350–650 nm. Sample concentration effects on ORD spectra of R-3MCH were also recorded and investigated over widely varying concentrations from 2.5 × 10−3 to 2.5 × 10−1 g/mL where an observed sensitivity of optical rotation (OR) to incident light wavelength at low concentrations is correlated to solvent effects. Temperature effects were also studied by recording ORD spectra over the temperature range 0–65 °C in toluene. Recorded specific OR was plotted against various solvent parameters, namely, dipole moment, polarity, refractive index and polarizability to probe solvent effects. Furthermore, solvent effects were studied by incorporating Kamlet's and Taft's solvent parameters in the multi-parametric linear fitting. Theoretically, ORD spectra and populations of optimized geometries of equatorial and axial conformers of R-3MCH were calculated in the gas and solvated phases. All theoretical calculations were performed employing the polarizable continuum model using density functional theoretical and composite scheme (G4) methods with aug-cc-pVTZ and aug-cc-pVDZ basis sets. Net ORD spectra of R-3MCH were generated by the Boltzmann-weighted sum of the contributions of the dominant conformers. Upon comparing theoretical and experimental ORD spectra, a very good agreement is observed for the ORD spectra in the gas phase and high polarity solvents compared to relatively lesser agreement in low polarity solvents.

Racemization is the key step to turn a kinetic resolution (KR), which suffers from the well-known drawback of being limited to a maximum yield of 50% with high enantiopurity, into a dynamic kinetic resolution (DKR) process. Enzyme-based racemization of enantiopure alcohols and amines has gained significant interest in recent years. This review covers recent advances in enzyme-based racemization approaches and their potential applications in bi-enzymatic DKR.

The exploitation of petroleum oil generates a considerable amount of “produced water or petroleum waste effluent (PWE)” that is contaminated with polycyclic aromatic hydrocarbons (PAHs), including Benzo[a]pyrene (BaP). PWE is characterised by its high salinity, which can be as high as 30% NaCl, thus the exploitation of biodegradation to remove PAHs necessitates the use of active halophilic microbes. The strain 10SBZ1A was isolated from oil contaminated soils, by enrichment experiment in medium containing 10% NaCl (w/v). Homology analyses of 16S rRNA sequences identified 10SBZ1A as a Staphylococcus haemoliticus species, based on 99.99% homology (NCBI, accession number GI: MN388897). The strain could grow in the presence of 4–200 μmol l-1 of BaP as the sole source of carbon, with a doubling time of 17–42 h. This strain optimum conditions for growth were 37 oC, 10% NaCl (w/v) and pH 7, and under these conditions, it degraded BaP at a rate of 0.8 μmol l-1 per day. The strain 10SBZ1A actively degraded PAHs of lower molecular weights than that of BaP, including pyrene, phenanthrene, anthracene. This strain was also capable of removing 80% of BaP in the context of soil spiked with BaP (10 μmol l-1 in 100 g of soil) within 30 days. Finally, a metabolic pathway of BaP was proposed, based on the identified metabolites using liquid chromatography-high resolution tandem mass spectrometry. To the best of our knowledge, this is the first report of a halophilic BaP degrading bacterial strain at salinity > 5% NaCl.

Bioremediation is based on microorganisms able to use pollutants either as a source of carbon or in co-metabolism, and is a promising strategy in cleaning the environment. Using soil contaminated with petroleum products from an industrial area in Saudi Arabia (Jubail), and after enrichment with the polycyclic aromatic hydrocarbon (PAH) naphthalene, a Methylobacterium radiotolerans strain (N7A0) was isolated that can grow in the presence of naphthalene as the sole source of carbon. M. radiotolerans is known to be resistant to gamma radiation, and this is the first documented report of a strain of this bacterium using a PAH as the sole source of carbon. The commonly reported Pseudomonas aeruginosa (strain N7B1) that biodegrades naphthalene was also identified, and gas chromatography analyses have shown that the biodegradation of naphthalene by M. radiotolerans and P. aeruginosa did follow both the salicylate and phthalate pathways.

We developed a single-enzyme-mediated two-step approach for deracemization of secondary alcohols. A single mutant of Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase enables the nonstereoselective oxidation of racemic alcohols to ketones, followed by a stereoselective reduction process. Varying the amounts of acetone and 2-propanol cosubstrates controls the stereoselectivities of the consecutive oxidation and reduction reactions, respectively. We used one enzyme to accomplish the deracemization of secondary alcohols with up to >99 % ee and >99.5 % recovery in one pot and without the need to isolate the prochiral ketone intermediate.

The immobilization of Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase (TeSADH) using sol–gel method enables its use to racemize enantiopure alcohols in organic media. Here, we report the racemization of enantiopure phenyl-ring-containing secondary alcohols using xerogel-immobilized W110A TeSADH in hexane rather than the aqueous medium required by the enzyme. We further showed that this racemization approach in organic solvent was compatible with Candida antarctica lipase B (CALB)-catalyzed kinetic resolution. This compatibility, therefore, allowed a dual enzymatic dynamic kinetic resolution of racemic alcohols using CALB-catalyzed kinetic resolution and W110A TeSADH-catalyzed racemization of phenyl-ring-containing alcohols.

Controlled racemization of enantiopure alcohols is a key step in dynamic kinetic resolution. We recently reported the racemization of enantiopure phenyl-ring-containing alcohols using W110A Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase (W110A TeSADH), which relies on selectivity mistakes. Trp-110 lines the large pocket of the active site of TeSADH, which allows W110A TeSADH mutant to accommodate phenyl-ring-containing substrates in Prelog mode, albeit with selectivity mistakes. Here, we report the racemization of enantiopure phenyl-ring-containing alcohols using several Trp-110 mutants of TeSADH in the presence of the oxidized and reduced forms of nicotinamide–adenine dinucleotide. We observed a noticeable enhancement in racemization efficiency when W110G TeSADH was used compared with W110Q, W110M, W110L, W110I, and W110V. This observation was anticipated because the W110G mutation is expected to open the large pocket of the active site to a greater extent compared to other mutants of TeSADH at W110. Both enantiomers of 1-phenyl-2-propanol and 4-phenyl-2-butanol were fully racemized by W110G TeSADH. We also constructed a triple mutant of TeSADH, W110A/I86A/C295A, by site-directed mutagenesis with the aim of opening the two pockets of the active site of TeSADH. The W110A/I86A/C295A mutant was employed to racemize enantiopure phenyl-ring-containing alcohols. The current study demonstrates that W110G and W110A/I86A/C295A TeSADH are more efficient catalysts for the racemization of enantiopure secondary alcohols than the previously reported mutant W110A TeSADH [6] .

The ability to control the substrate specificity and stereochemistry of enzymatic reactions is of increasing interest in biocatalysis. As this review highlights, this control can be achieved throug...

Alcohol dehydrogenases (ADHs) are enzymes that catalyze the reversible reduction of carbonyl compounds to their corresponding alcohols. We have been studying a thermostable, nicotinamide-adenine dinucleotide phosphate (NADP+)-dependent, secondary ADH from Thermoanaerobacter ethanolicus (TeSADH). In the current work, we expanded our library of TeSADH and adopted the site-saturation mutagenesis approach in creating a comprehensive mutant library at W110. We used phenylacetone as a model substrate to study the effectiveness of our library because this substrate showed low enantioselectivity in our previous work when reduced using W110A TeSADH. Five of the newly designed W110 mutants reduced phenylacetone at >99.9% ee, and two of these mutants exhibit an enantiomeric ratio (E-value) of over 100. These five mutants also reduced 1-phenyl-2-butanone and 4-phenyl-2-butanone to their corresponding (S)-configured alcohols in >99.9% ee. These new mutants of TeSADH will likely have synthetic utility for reduction of aromatic ketones in the future.

The cAMP-dependent protein kinase [protein kinase A (PKA)] mediates a myriad of cellular signaling events, and its activity is tightly regulated in both space and time. Among these regulatory mechanisms is N-myristoylation, whose biological role has been elusive. Using a combination of thermodynamics, kinetics, and spectroscopic methods, we analyzed the effects of N-myristoylation and phosphorylation at Ser10 on the interactions of PKA with model membranes. We found that, in the absence of lipids, the myristoyl group is tucked into the hydrophobic binding pocket of the enzyme (myr-in state). Upon association with lipid bilayers, the myristoyl group is extruded and inserts into the hydrocarbon region of the lipid bilayer (myr-out state). NMR data indicate that the enzyme undergoes conformational equilibrium between myr-in and myr-out states, which can be shifted byeither interaction with membranes and/or phosphorylation at Ser10. Our results provide evidence that the membrane binding motif of the myristoylated C-subunit of PKA (PKA-C) steers the enzyme toward lipids independent of its regulatory subunit or an A-kinase anchoring protein, providing an additional mechanism to localize the enzyme near membrane-bound substrates.

Reactions of M(CO)5THF, which was generated photochemically from M(CO)6 in THF, with (CH3)3CS(CH2)nSC(CH3)3 (=SS; n=5, 6; M=W, Cr) at room temperature afforded exclusively the bimetallic complexes (CO)5MSSM(CO)5. These new complexes with dithiaalkane bridging ligands have been characterized by IR and 1H NMR spectroscopies and elemental analysis. Kinetic studies of ligand-exchange reactions in these complexes in chlorobenzene (=CB) solutions employing tri(iso-propyl)phosphite (=L) as an incoming nucleophile indicated that the bridging ligand SS is replaced by L to afford finally (CO)5MP(O-i-Pr)3 as the sole reaction product. The kinetic data have also confirmed that replacement of SS by L under pseudo-first-order reaction conditions is dissociative and proceeds via a mechanism which involves initial M-S bond breaking followed by other steps. First order rate constants and activation parameters for these reactions have been determined.

The efficiency of biocatalytic redox reactions catalyzed by alcohol dehydrogenases (ADHs) have been the subject of considerable research recently. Two major challenges have restricted their application in asymmetric synthesis until now. First of all, most of the interesting substrates are either insoluble or sparingly soluble in aqueous media, the natural medium for enzymes. This drawback has been overcome by using non-aqueous media like organic solvents, ionic liquids, and supercritical carbon dioxide in mono- and biphasic reactions, and several ADHs show high activity at high concentrations of such reaction media. The second challenge is the strict substrate specificity for most ADHs. The continuous search for new ADHs, together with random and rational mutagenesis to widen substrate specificity, will help in attracting organic chemists to consider utilizing them in organic synthesis more often. The aim of this perspective is to highlight recent efforts to overcome the above-mentioned limitations.

Reactions of the intermediate W(CO)5THF, generated photochemically from W(CO)6 in THF, with Ph2P(CH2) n PPh2 [ = PP; n = 2 (dppe), 4 (dppb), 6 (dpph), 10 (dppd)] at room temperature in THF solutions gave exclusively bimetallic complexes of the (CO)5WPPW(CO)5 type. In addition, complexes bridged by diphosphine ligands of the (CO)4(pip)MPPM(pip)(CO)4 type (pip = piperidine; M = Mo, W) were prepared by stirring the (CO)4M(pip)2 complexes with bis(diphenylphosphino)alkanes in CH2Cl2 solution at ambient temperatures. These new bis(diphenylphosphino)alkane-bridged complexes were characterized by i.r., 1H- and 31P-n.m.r. spectroscopies, as well as by elemental analysis.

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.

Theasymmetricreductionofketonesandthekinetic resolution (KR)of racemic alcohols are the mostimportant reactions for producing optically active alcoholsthat then can be used to synthesize industrially importantcompounds like natural products.Apractical technique toimprove enzyme performanceisenzyme immobilization.

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.

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).

Controlled racemization of enantiopure phenyl-ring-containing secondary alcohols is achieved in this study using W110A secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus (W110A TeSADH) and in the presence of the reduced and oxidized forms of its cofactor nicotinamide-adenine dinucleotide. Racemization of both enantiomers of alcohols accepted by W110A TeSADH, not only with low, but also with reasonably high, enantiomeric discrimination is achieved by this method. Furthermore, the high tolerance of TeSADH to organic solvents allows TeSADH-catalyzed racemization to be conducted in media containing up to 50% (v/v) of organic solvents.

The efficiency of biocatalytic redox reactions catalyzed by alcohol dehydrogenases (ADHs) have been the subject of considerable research recently. Two major challenges have restricted their application in asymmetric synthesis until now. First of all, most of the interesting substrates are either insoluble or sparingly soluble in aqueous media, the natural medium for enzymes. This drawback has been overcome by using non-aqueous media like organic solvents, ionic liquids, and supercritical carbon dioxide in mono- and biphasic reactions, and several ADHs show high activity at high concentrations of such reaction media. The second challenge is the strict substrate specificity for most ADHs. The continuous search for new ADHs, together with random and rational mutagenesis to widen substrate specificity, will help in attracting organic chemists to consider utilizing them in organic synthesis more often. The aim of this perspective is to highlight recent efforts to overcome the above-mentioned limitations.

The catalytic subunit of cAMP-dependent protein kinase A (PKA-C), controls the activation and deactivation of over 100 unique cytosolic and membrane bound substrates. Nearly all the PKA-C in mammals is subjected to myristoylation and myristoylation can be dependent on cell/tissue type. Although the function of myristoylation is not known, it has been hypothesized to mediate PKA-C/membrane and PKA-C/membrane protein interactions. Here, we developed an in vitro myristoylation method to obtain homogeneously myristoylated PKA-C (myrPKA-C) in quantities suitable for NMR studies. Using NMR spectroscopy, the interactions of membrane mimicking isotropic bicelles (DMPC/DHPC) with 15N-labeled myrPKA-C or 15N-labeled non-myristoylated PKA-C was monitored by [1H-15N]-TROSY-HSQC spectroscopy. Significant chemical shift perturbations were detected for the N-terminus and the myristate pocket of myrPKA-C, while both perturbations and line broadening was detected in remote regions far away from the myristoyl binding pocket. Such changes were not observed for the non-myristoylated form, suggesting that myristoylation drives the enzyme towards the membrane and may act allosterically on the enzyme. Membrane localization was further supported by intrinsic tryptophan fluorescence measurements which showed drastically different patterns between myrPKA-C and PKA-C. Finally, the insertion of the myristate group into lipid membranes was confirmed using 2H solid-state NMR spectroscopy on myrPKA-C with a deuterated myristoyl group. We propose that the myristoyl group of myrPKA-C steers the enzyme towards the membrane, even in the absence of its regulatory subunit or A-kinase anchoring proteins (AKAPs). These results provide an additional mechanism for PKA localization and recognition of membrane bound substrates. Preliminary data on PKA recognition of the integral membrane protein, phospholamban, are also presented.