Substrate promiscuity in evolved Alcohol Dehydrogenase A (ADH-A)

Alcohol dehydrogenase A (ADH-A) from Rhodococcus ruber DSM 44541 tolerates organic solvents, and therefore this became a useful biocatalyst for asymmetric synthesis of organic compounds.1 ADH-A is capable of catalyzing stereoselective oxidation of phenyl-substituted sec-alcohols and reduction of the corresponding ketones. Importantly, these compounds are precursors for the synthesis of a range of biologically active compounds.1,2 Therefore, we have been studying engineering of ADH-A for the purpose of developing new enzymes with pre-designed catalytic properties regarding substrate scope and selectivity. We have been isolated a number of ADH-A variants which have been isolated from CASTing libraries for different purposes and function. Variants isolated from a library originally generated from random mutagenesis of residues Y294 and W295 (called “A”, clones A1, A2, A2C3 and A2C2B1) represent hits from different generation of directed evolution, selected for improved activity for the non-preferred R-enantiomer of 1-phenylethanol.2 Other mutants that were selected (variants B1 and B1F4) for improved activity with a disubstituted sec-alcohol also displayed altered regioselectivity as compared to the wild type.3 In a third evolution effort, enzyme variants C1 and C1B1 were isolated after selection for improved activity with the vicinal diol (R)-1-pheny-1,2-ethanediol.4 Please click Additional Files below to see the full abstract

The angle of a side-chain decides regio- and enantioselectivity in Alcohol Dehydrogenase A

Alcohol dehydrogenase A (ADH-A) from Rhodococcus ruber DSM 44541 is a promising biocatalyst for asymmetric synthesis of organic compounds.1 This enzyme is capable of catalyzing enantio- and regioselectivity of phenyl-substitute a-hydroxy ketones (acyloins), which are precursors for the synthesis of a range of biologically active compounds.1,2 ADH-A catalyzes the oxidation of (S)-1-phenylethanol 3000-fold more efficiently as compared to the 2-hydroxylated derivatives (R)-phenyl-1,2-ethanediol. ADH-A is highly selective towards secondary-alcohols and displays very low activities with corresponding primary-alcohol derivatives.2,3 Apparently, when this selectivity was tested with substrate contained two secondary-alcohols, we analyzed the catalytic efficiency and the regioselectivity towards (1R,2S)-2.2 The conclusions were yielded that ADH-A is a comparably inefficient catalyst for oxidation of vicinal diols, but displays regioselectivity, oxidizing primarily the benzylic carbon of this substrate.2 Please click Additional Files below to see the full abstract

Редокс-регуляция динамического характера фенотипа эндотелиоцитов кровеносных сосудов

ЭНДОТЕЛИАЛЬНЫЕ КЛЕТКИКЛЕТКИЭНДОТЕЛИЙКРОВЕНОСНЫЕ СОСУДЫФЕНОТИПГЕНЕТИЧЕСКИЕ ФЕНОМЕНЫОКИСЛИТЕЛЬНО-ВОССТАНОВИТЕЛЬНЫЕ РЕАКЦИИЭНЕРГЕТИЧЕСКИЙ ОБМЕНПЕРЕКИСНОЕ ОКИСЛЕНИЕ ЛИПИДО

New Thermophilic α/β Class Epoxide Hydrolases Found in Metagenomes From Hot Environments

This is the final version. Available from Frontiers Media via the DOI in this record.Two novel epoxide hydrolases (EHs), Sibe-EH and CH65-EH, were identified in the metagenomes of samples collected in hot springs in Russia and China, respectively. The two α/β hydrolase superfamily fold enzymes were cloned, over-expressed in Escherichia coli, purified and characterized. The new EHs were active toward a broad range of substrates, and in particular, Sibe-EH was excellent in the desymmetrization of cis-2,3-epoxybutane producing the (2R,3R)-diol product with ee exceeding 99%. Interestingly these enzymes also hydrolyse (4R)-limonene-1,2-epoxide with Sibe-EH being specific for the trans isomer. The Sibe-EH is a monomer in solution whereas the CH65-EH is a dimer. Both enzymes showed high melting temperatures with the CH65-EH being the highest at 85°C retaining 80% of its initial activity after 3 h thermal treatment at 70°C making it the most thermal tolerant wild type epoxide hydrolase described. The Sibe-EH and CH65-EH have been crystallized and their structures determined to high resolution, 1.6 and 1.4 Å, respectively. The CH65-EH enzyme forms a dimer via its cap domains with different relative orientation of the monomers compared to previously described EHs. The entrance to the active site cavity is located in a different position in CH65-EH and Sibe-EH in relation to other known bacterial and mammalian EHs