Exploiting Substrate Promiscuity of Ectoine Hydroxylase for Regio- and Stereoselective Modification of Homoectoine.

Extant enzymes are not only highly efficient biocatalysts for a single, or a group of chemically closely related substrates but often have retained, as a mark of their evolutionary history, a certain degree of substrate ambiguity. We have exploited the substrate ambiguity of the ectoine hydroxylase (EctD), a member of the non-heme Fe(II)-containing and 2-oxoglutarate-dependent dioxygenase superfamily, for such a task. Naturally, the EctD enzyme performs a precise regio- and stereoselective hydroxylation of the ubiquitous stress protectant and chemical chaperone ectoine (possessing a six-membered pyrimidine ring structure) to yield -5-hydroxyectoine. Using a synthetic ectoine derivative, homoectoine, which possesses an expanded seven-membered diazepine ring structure, we were able to selectively generate, both and , -5-hydroxyhomoectoine. For this transformation, we specifically used the EctD enzyme from in a whole cell biocatalyst approach, as this enzyme exhibits high catalytic efficiency not only for its natural substrate ectoine but also for homoectoine. Molecular docking approaches with the crystal structure of the EctD protein predicted the formation of -5-hydroxyhomoectoine, a stereochemical configuration that we experimentally verified by nuclear-magnetic resonance spectroscopy. An cell factory expressing the gene from a synthetic promoter imported homoectoine the ProU and ProP compatible solute transporters, hydroxylated it, and secreted the formed -5-hydroxyhomoectoine, independent from all currently known mechanosensitive channels, into the growth medium from which it could be purified by high-pressure liquid chromatography.