Exploring the substrate scope of glycerol dehydrogenase GldA from E. coli BW25113 towards cis-dihydrocatechol derivatives.

Glycerol dehydrogenase (GldA) from Escherichia coli BW25113, naturally catalyzes the oxidation of glycerol to dihydroxyacetone. It is known that GldA exhibits promiscuity towards short-chain C2-C4 alcohols. However, there are no reports regarding the substrate scope of GldA towards larger substrates. Herein we demonstrate that GldA can accept bulkier C6-C8 alcohols than previously anticipated. Overexpression of the gldA gene in the knockout background, E. coli BW25113 ΔgldA, was strikingly effective converting 2 mM of the compounds: cis-dihydrocatetechol, cis-(1 S,2 R)- 3-methylcyclohexa-3,5-diene-1,2-diol and cis-(1 S,2 R)- 3-ethylcyclohexa-3,5-diene-1,2-diol, into 2.04 ± 0.21 mM of catechol, 0.62 ± 0.11 mM 3-methylcatechol, and 0.16 ± 0.02 mM 3-ethylcatechol, respectively. In-silico studies on the active site of GldA enlightened the decrease in product formation as the steric substrate demand increased. These results are of high interests for E. coli-based cell factories expressing Rieske non-heme iron dioxygenases, producing cis-dihydrocatechols, since such sough-after valuable products can be immediately degraded by GldA, substantially hampering the expected performance of the recombinant platform.

C-H Amination via Nitrene Transfer Catalyzed by Mononuclear Non-Heme Iron-Dependent Enzymes.

Expanding the reaction scope of natural metalloenzymes can provide new opportunities for biocatalysis. Mononuclear non-heme iron-dependent enzymes represent a large class of biological catalysts involved in the biosynthesis of natural products and catabolism of xenobiotics, among other processes. Here, we report that several members of this enzyme family, including Rieske dioxygenases as well as α-ketoglutarate-dependent dioxygenases and halogenases, are able to catalyze the intramolecular C-H amination of a sulfonyl azide substrate, thereby exhibiting a promiscuous nitrene transfer reactivity. One of these enzymes, naphthalene dioxygenase (NDO), was further engineered resulting in several active site variants that function as C-H aminases. Furthermore, this enzyme could be applied to execute this non-native transformation on a gram scale in a bioreactor, thus demonstrating its potential for synthetic applications. These studies highlight the functional versatility of non-heme iron-dependent enzymes and pave the way to their further investigation and development as promising biocatalysts for non-native metal-catalyzed transformations.

Toluene Dioxygenase-Catalysed Oxidation of Benzyl Azide to Benzonitrile: Mechanistic Insights for an Unprecedented Enzymatic Transformation.

Enzymatic dioxygenation of benzyl azide by toluene dioxygenase (TDO) produces significant amounts of the cis-cyclohexadienediol derived from benzonitrile, along with the expected azido diols. We demonstrate that TDO catalyses the oxidation of benzyl azide to benzonitrile, which is further dioxygenated to produce the observed cis-diol. A proposed mechanism for this transformation involves initial benzylic monooxygenation followed by a nitrene-mediated rearrangement to form an oxime, which is further dehydrated to afford the nitrile. To the best of our knowledge, this is the first report of enzymatic oxidation of an alkyl azide to a nitrile. In addition, the described oxime-dehydration activity has not been reported for Rieske dioxygenases.

Double [3,3]-sigmatropic rearrangement in the enzymatic dioxygenation of benzyl azide: preparation of novel synthetically valuable azido-diols.

Enzymatic dioxygenation of benzyl azide by toluene dioxygenase produces the expected enantiopure cis-cyclohexadienediol along with an exocyclic diene formed by a spontaneous sequence of two [3,3] sigmatropic shifts. This novel dienediol presents high synthetic potential for natural product synthesis. The sigmatropic rearrangements can be reversed by protection of the diol moiety. An optimized production protocol for either of these valuable diols is presented.