Deracemization and Stereoinversion of Alcohols Using Two Mutants of Secondary Alcohol Dehydrogenase from Thermoanaerobacter pseudoethanolicus (original) (raw)
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European Journal of Organic Chemistry
Here, we report the asymmetric reduction of selected phenyl-ring-containing ketones by various single and dual site mutants of Thermoanaerobacter pseudoethanolicus secondary alcohol dehydrogenase (TeSADH). Further expanding the size of the substrate binding pocket in the mutant W110A/I86A not only allowed substrates of the single mutants W110A and I86A to be accommodated within the expanded active site, but also expanded the enzyme's substrate range to ketones bearing two sterically demanding groups (bulky-bulky ketones), which are not substrates for TeSADH single mutants. We also report the regio-and enantioselective reduction of diketones using W110A/I86A TeSADH and single TeSADH mutants. The double mutant exhibited dual stereopreference generating the Prelog products most of the time and the anti-Prelog products in a few cases.
Applied and environmental …, 1988
Thermoanaerobacter ethanolicus (ATCC 31550) has primary and secondary alcohol dehydrogenases. The two enzymes were purified to homogeneity as judged from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. The apparent MrS of the primary and secondary alcohol dehydrogenases are 184,000 and 172,000, respectively. Both enzymes have high thermostability. They are tetrameric with apparently identical subunits and contain from 3.2 to 5.5 atoms of Zn per subunit. The two dehydrogenases are NADP dependent and reversibly convert ethanol and 1-propanol to the respective aldehydes. The V1m values with ethanol as a substrate are 45.6 ,umol/min per mg for the primary alcohol dehydrogenase and 13 ,umol/min per mg for the secondary alcohol dehydrogenase at pH 8.9 and 60°C. The primary enzyme oxidizes primary alcohols, including up to heptanol, at rates similar to that of ethanol. It is inactive with secondary alcohols. The secondary enzyme is inactive with 1-pentanol or longer chain alcohols. Its best substrate is 2-propanol, which is oxidized 15 times faster than ethanol. The secondary alcohol dehydrogenase is formed early during the growth cycle. It is stimulated by pyruvate and has a low Km for acetaldehyde (44.8 mM) in comparison to that of the primary alcohol dehydrogenase (210 mM). The latter enzyme is formed late in the growth cycle. It is postulated that the secondary alcohol dehydrogenase is largely responsible for the formation of ethanol in fermentations of carbohydrates by T. ethanolicus.
Biochemical Journal, 1997
The Thermoanaerobacter ethanolicus 39E adhB gene encoding the secondary-alcohol dehydrogenase (2m ADH) was overexpressed in Escherichia coli at more than 10 % of total protein. The recombinant enzyme was purified in high yield (67 %) by heat-treatment at 85 mC and (NH %) # SO % precipitation. Sitedirected mutants (C37S, H59N, D150N, D150E and D150C were analysed to test the peptide sequence comparison-based predictions of amino acids responsible for putative catalytic Zn binding. X-ray absorption spectroscopy confirmed the presence of a protein-bound Zn atom with ZnS " (imid) " (N,O) $ coordination sphere. Inductively coupled plasma atomic emission spectrometry measured 0n48 Zn atoms per wild-type 2m ADH subunit. The C37S, H59N and D150N mutant enzymes bound only 0n11, 0n13 and 0n33 Zn per subunit respectively, suggesting that these residues are involved in Zn liganding. The D150E and D150C mutants retained 0n47 and 1n2 Zn atoms per subunit, indicating that an anionic side-chain moiety at this position preserves the bound Zn. All five mutant enzymes had 3 % of wild-type
Protein Engineering Design and Selection, 2007
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)-2butanol 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)-2butanol's orientation in the TbSADH †(S)-2-butanol binary complex does not reflect its orientation in the ternary enzyme -substrate-cofactor complex.
The Biochemical journal, 1997
The Thermoanaerobacter ethanolicus 39E adhB gene encoding the secondary-alcohol dehydrogenase (secondary ADH) was overexpressed in Escherichia coli at more than 10% of total protein. The recombinant enzyme was purified in high yield (67%) by heat-treatment at 85 degrees C and (NH4)2SO4 precipitation. Site-directed mutants (C37S, H59N, D150N, D150Eand D150C were analysed to test the peptide sequence comparison-based predictions of amino acids responsible for putative catalytic Zn binding. X-ray absorption spectroscopy confirmed the presence of a protein-bound Zn atom with ZnS1(imid)1(N,O)3 co-ordination sphere. Inductively coupled plasma atomic emission spectrometry measured 0.48 Zn atoms per wild-type secondary ADH subunit. The C37S, H59N and D150N mutant enzymes bound only 0.11, 0.13 and 0.33 Zn per subunit respectively,suggesting that these residues are involved in Zn liganding. The D150E and D150C mutants retained 0.47 and 1.2 Zn atoms per subunit, indicating that an anionic side...
Journal of Molecular Catalysis B: Enzymatic, 2015
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 .
Organic & Biomolecular Chemistry, 2013
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. † Electronic supplementary information (ESI) available: GC chromatograms for the racemization products. See