Thermoanaerobium Brockii Alcohol Dehydrogenase Research Articles

Immobilization of Thermoanaerobium brockii alcohol dehydrogenase on SBA-15

The activity of Thermoanaerobium brockii alcohol dehydrogenase (TBADH) adsorbed on mesoporous silica SBA-15 was compared with that of the free enzyme in water and in biphasic system (water phase up to 50% v/v water). TBADH was active at a water concentration ≥10% v/v. In the reduction reaction of sulcatone to sulcatol carried out in biphasic systems, the yield obtained with SBA-15-adsorbed TBADH was up to 5.5-fold higher than that with the free enzyme, which suggests a higher stability of the immobilized enzyme toward the organic solvent. The nature of the organic solvent substantially influenced the degree of conversion that, for example, was 7.4% in toluene and 31.6% in petroleum ether.

Microscale synthesis and kinetic isotope effect analysis of (4R)-[Ad-14C, 4-2H] NADPH and (4R)-[Ad-3H,4-2H] NADPH.

We present a one-pot chemo-enzymatic microscale synthesis of NADPH with two different patterns of isotopic labels: (4R)-[Ad-14C,4-2H] NADPH and (4R)-[Ad-3H,4-2H] NADPH. These co-factors are required by an enormous range of enzymes, and isotopically labeled nicotinamides are consequently of significant interest to researchers. In the current procedure, [Ad-14C] NAD+ and [Ad-3H] NAD+ were phosphorylated by NAD+ kinase to produce [Ad-14C] NADP+ and [Ad-3H] NADP+, respectively. Thermoanaerobium brockii alcohol dehydrogenase was then used to stereospecifically transfer deuterium from C2 of isopropanol to the re face of C4 of NADP+. After purification by HPLC, NMR analysis indicated the deuterium content at the 4R position is more than 99.7 %. The labeled cofactors were then used to successfully and sensitively measure kinetic isotope effects for R67 dihydrofolate reductase, providing strong evidence for the utility of this synthetic methodology.

Microscale synthesis of isotopically labeled 6R‐N5, N10 methylene‐5, 6, 7, 8‐tetrahydrofolate

AbstractA one‐pot chemo‐enzymatic microscale synthesis of isotopically labeled R‐[6‐YH; 11‐XH] N5, N10 methylene‐5, 6, 7, 8‐tetrahydrofolate (CH2H4folate) is presented, where Y=1 or 2 represents protium or deuterium, and X=1, 2 or 3 represents protium, deuterium or tritium, respectively. In this procedure, Thermoanaerobium brockii alcohol dehydrogenase (tbADH) and Escherichia coli dihydrofolate reductase (ecDHFR) were used simultaneously in the reaction mixture. First, tbADH stereospecifically catalyzes a hydride transfer from [2‐YH] iPrOH to the re face of C‐4 NADP+. The ecDHFR then reduced 7, 8‐dihydrofolate (H2folate) to form (6S)‐H4folate. Finally, the enzymatic reactions were followed by chemical trapping with isotopically labeled formaldehyde ([XH]‐HCHO) to form the final product. The preparation of deuterium‐ and tritium‐labeled formaldehyde is also presented. Two reverse phase HPLC methods were developed for analysis and purification of product R‐[6‐YH; 11‐XH] CH2H4folate. This isotopically labeled cofactor can be used to study 1° and 2° kinetic isotope effects (KIEs) with any CH2H4folate dependent enzyme as demonstrated by studies with E. coli thymidylate synthase (TS). Copyright © 2005 John Wiley & Sons, Ltd.

TBADH activity in water-miscible organic solvents: correlations between enzyme performance, enantioselectivity and protein structure through spectroscopic studies

The enantioselective reduction of 2-pentanone to (R)- and (S)-2-pentanol by Thermoanaerobacter (formerly Thermoanaerobium) brockii alcohol dehydrogenase (TBADH) in mixtures of water and water-miscible organic solvents was investigated. Significant enzymatic activity was retained in up to 87% methanol, ethanol and acetonitrile. The changes in enzyme activity as a function of organic solvent were correlated to structural alterations of TBADH with a series of spectroscopic studies (fluorescence, fluorescence quenching and circular dichroism (CD)). Interestingly, this study shows that the tetrameric form of TBADH is not critical for catalytic performance.

Synthesis of pyridine nucleotide analogs consisting of nicotinoylamino acids by means of transglycosidation reactions catalyzed by mammalian pyridine nucleotide transglycosidases.

Besides isonicotinic acid analogs of pyridine nucleotides, 24 novel pyridine nucleotide cofactors that have an amino acid residue at the carbonyl carbon of the nicotinamide moiety have been prepared by means of transglycosidation reactions catalyzed by rabbit spleen and guinea pig spleen pyridine nucleotide transglycosidases. Their chemical properties were characterized by means of proton NMR, Fab-mass, and UV spectral measurement and phosphodiesterase digestion. Except for the isonicotinic acid ones, these nicotinoylamino acid analogs were shown to function as substrates for both the hydrolysis and the transglycosidation reactions catalyzed by the mammalian NAD glycohydrolases, though their substrate activities were lower than those with the original pyridine nucleotides (NMN, NAD, and NADP). They were inactive in regard to yeast alcohol dehydrogenase- and Thermoanaerobium brockii alcohol dehydrogenase (NADP dependent)-oxidation.

Total Synthesis of (+)-Aspicilin. The Naked Carbon Skeleton Strategy vs the Bioorganic Approach.

The advantages of the "naked carbon skeleton" strategy in the total synthesis of polyoxygenated natural products are demonstrated in the total synthesis of the 18-membered macrolide (+)-aspicilin, 1. This approach employs the easily prepared, nonfunctionalized carbon skeleton of the target molecule, hexadeca-1,3,15-triene, 2. All the required stereogenic carbinol centers are then introduced onto this partially unsaturated hydrocarbon chain using the Sharpless asymmetric dihydroxylation (AD) reaction. Thus, asymmetric synthesis of 1 is achieved in 14 steps and 11% overall yield. Three stereogenic carbinol centers are introduced with very high regio- and enantioselectivity (epimeric excess of 96% at positions 3, 4, and 86% at position 15) by using AD-mix-beta while the fourth is obtained using AD-mix-alpha. This approach is compared with an alternative synthesis of 1 (19 steps and 4.3% overall yield) using chiral building blocks derived from D-arabinose and from the enzymatic reduction of oct-7-yn-2-one, 34, with Thermoanaerobium brockii alcoholdehydrogenase (TBADH).

Steady-state kinetic mechanism of Thermoanaerobium brockii alcohol dehydrogenase: A study of discrimination between alternative kinetic models

Two steady-state kinetic mechanisms have been proposed for the oxidation of 2-propanol catalyzed by Thermoanaerobium brockii alcohol dehydrogenase (TBADH): the sequential ordered BiBi mechanism and the Theorell-Chance BiBi mechanism. Discrimination between these kinetic mechanisms was achieved by analyzing the inhibition produced by acetone, the first product of the reaction sequence in relation to the second substrate of the enzyme (2-propanol). Two types of experimental data were used: a data set combining initial rate experiments in the absence of products with data involving inhibition by acetone in relation to 2-propanol as variable substrate; and, data sets just containing product inhibition experiments by acetone with 2-propanol as variable substrate. The results obtained strongly suggest that the kinetic mechanism of TBADH is described by the Theorell-Chance BiBi kinetic model.

Kinetic mechanism of the oxidation of 2-propanol catalyzed by Thermoanaerobium brockii alcohol dehydrogenase

The reaction of oxidation of 2-propanol to acetone in the presence of NADP catalyzed by Thermoanaerobium brockii alcohol dehydrogenase (TBADH) has been studied for gaining insight into the coupled-substrate approach that has been extensively used for NADPH regeneration in preparative-scale reductions catalyzed by this enzyme. The data obtained in initial velocity experiments in the absence of products as well as those involving product inhibition and dead-end inhibition by pyrazole suggest that the reaction mechanism is of the Theorell-Chance BiBi type and in which the coenzymes bind to the free form of the enzyme. The seven kinetic parameters that characterize this kinetic model were estimated by non-linear regression analysis using the appropriate rate equations. The kinetic mechanism proposed in the present work is fully compatible with the coupled-substrate approach used for regeneration of NADPH in reduction reactions catalyzed by TBADH.

Stereospecificity of hydrogen transfer by the NADP‐linked alcohol dehydrogenase from the thermophilic bacterium Thermoanaerobium brockii

Class A and class B NAD(H)/NADP(H) coenzyme-dependent dehydrogenases distinguish between the diastereotopic hydrogens pro-R and pro-S at position 4 of the cofactor. We investigated the stereochemistry of hydride transfer in reactions catalyzed by an unusual thermophilic, zinc-containing, NADP-linked enzyme Thermoanaerobium brockii alcohol dehydrogenase (TBAD). Using proton NMR spectroscopy of monodeuterated alcohols and coenzymes we found that TBAD is a class A enzyme that transfers the pro-R hydrogen from the pyridine 4 position of the reduced coenzyme. This stereospecificity is stable over (a) a broad range of temperatures up to 70 degrees C, (b) different concentrations of the coenzyme (catalytic or stoichiometric) and (c) a wide scope of substrates. Although NAD+ is not an effective coenzyme for TBAD, NADP+ and its synthetic analogs, 3-acetylpyridine-ADP+ and thio-NADP+, can be used successfully.

Thermostable enzymes in organic synthesis. 7. Total synthesis of the western corn rootworm sex pheromone 8-methyldec-2-yl propanoate using a TBADH-generated C2-bifunctional chiron

Enantiomerically pure alcohols produced by Thermoanaerobium brockii alcohol dehydrogenase (TBADH)catalyzed asymmetric reduction of polyfunctional ketones are useful building blocks for natural products synthesis. This advantage has been demonstrated by a total synthesis of all four isomers of 8-methyldec-2-yl propanoate, the sex pheromone emitted by the female western corn rootworm, Diabrotica virgifera virgifera LeConte. These four isomers were obtained in a very short procedure (either three or five steps) with an enantiomeric purity that exceeds 99% using a single chiral building block

Thermostable enzymes in organic synthesis, Part 6. Total synthesis of (S)–(–)-zearalenone using a TBADH-generated trifunctional chiron

Chiral alcohols produced by Thermoanaerobium brockii alcohol dehydrogenase (TBADH)-catalysed asymmetric reduction of polyfunctional ketones are useful building blocks for natural products synthesis. In particular, the ability of TBADH to discriminate between two ketones having equal chemical reactivity is demonstrated by enzymatic reduction of dec-9-ene-2, 6-dione to produce optically pure (S)-2-hydroxydec-9-en-6-one. The total synthesis of (S)-(–)-zearalenone with optical purity that exceeds 99.5% has been achieved by using the latter compound as a starting material.

Thermostable enzymes in organic synthesis, 5. Total synthesis of S-(+)-Z-dodec-3-en-11-olide (ferrulactone II) using a tbadh-generated bifunctional chiron.

The total synthesis of (S)-(+)-ferrulactone ( 2), one of several synergistic aggregation pheromones produced by male flat grain beetles, Cryptolestes ferrugineus (Stephens) was achieved in a five-step synthesis in very high optical purity (>99% ee) and 17.5% chemical yield, starting from (S)-(+)-methyl-8-hydroxynonanoate ( 5) and a three-carbon Wittig reagent 11. The nine-carbon bifunctional chiron 5 was obtained from methyl 8-oxononanoate via enantioselective reduction with Thermoanaerobium brockii alcohol dehydrogenase (TBADH).

Unusual stereoselectivity in the reduction of bicyclo[3.3.0]oct-2-en-8-one by Thermoanaerobiumbrockii alcohol dehydrogenase

The Thermoanaerobium brockii alcohol dehydrogenase (TBADH) catalysed reduction of the racemic bicyclic ketone 5 gives epimeric alcohols derived from the same enantiomeric series, the reduction is enantioselective but not diastereoselective, an unusual result for a single enzyme.

Thermostable Enzymes in Organic Synthesis, 4. Tbadh-Catalyzed Preparation of Bifunctional Chirons. Total Synthesis of S-(+)-Z-Tetradec-5-En-13-Olide

Highly enantioselective reduction of various methyl- and ethylketones bearing different functional groups, such as double and triple carbon-carbon bonds, methyl ester, cyano, ethyl ether, phenyl and chloride, employing Thermoanaerobium brockii alcohol dehydrogenase (TBADH) as a catalyst, affords the corresponding optically active, secondary alcohols. As expected on the basis of our previous studies with monofunctional ketones, reduction of most of the substrates yields, uniformly, alcohols with an S configuration, arising from highly selective hydride attack at the re face of the carbonyl. However, with the smaller-sized ketones, there is a clear reversal in stereoselectivity. The synthetic usefulness of these chiral building blocks has been demonstrated by the total synthesis of (S)-(+)-Z-tetradec-5-en-13-olide, one of several synergistic aggregation pheromones produced by male flat grain beetles, Cryptolestes pusillus (Schonherr). The pheromone was prepared from (S)-(+)-methyl-8-hydroxynonanoate with optical purity greater than 99% in a six-step synthesis.

ChemInform Abstract: Organic Synthesis with Enzymes. Part 3. TBADH‐Catalyzed Reduction of Chloro Ketones.

AbstractHighly enantioselective reduction of the chloro ketones (I) is achieved by TBADH (Thermoanaerobium brockii alcohol dehydrogenase) reduction to afford the alcohols (II).