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

Abstract

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 trans-5-hydroxyectoine. Using a synthetic ectoine derivative, homoectoine, which possesses an expanded seven-membered diazepine ring structure, we were able to selectively generate, both in vitro and in vivo, trans-5-hydroxyhomoectoine. For this transformation, we specifically used the EctD enzyme from Pseudomonas stutzeri 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 Sphingopyxis alaskensis EctD protein predicted the formation of trans-5-hydroxyhomoectoine, a stereochemical configuration that we experimentally verified by nuclear-magnetic resonance spectroscopy. An Escherichia coli cell factory expressing the P. stutzeri ectD gene from a synthetic promoter imported homoectoine via the ProU and ProP compatible solute transporters, hydroxylated it, and secreted the formed trans-5-hydroxyhomoectoine, independent from all currently known mechanosensitive channels, into the growth medium from which it could be purified by high-pressure liquid chromatography.

Prospective CO2 and CO bioconversion into ectoines using novel microbial platforms

“…1): l-2,4-diaminobutyric acid (DABA) transaminase (EctB), DABA acetyltransferase (EctA) and ectoine synthase (EctC). EctC is considered a marker protein for ectoine producers (Czech et al 2019). Sometimes an additional gene of…”

“…A compatible solute derived from ectoine that has recently received commercial attention is hydroxyectoine (5-hydroxy-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid) (Czech et al 2019). Hydroxyectoine is produced by a substantial amount of ectoine producers through a positionand stereo-specific hydroxylation catalyzed by an ectoine hydroxylase (EctD) (Czech et al 2018a).…”

The ups and downs of ectoine: structural enzymology of a major microbial stress protectant and versatile nutrient

“…So far, nine ectoine hydroxylases have been characterized biochemically, including enzymes from various extremophilic Bacteria and also one protein from a marine archaeon (Nitrosopumilus maritimus) (Bursy et al 2007;Reuter et al 2010;Widderich et al 2014aWidderich et al , 2016a. These enzymes seemed to possess similar kinetic parameters, but the subsequent optimization of assay conditions for individual enzymes revealed significant differences in their catalytic efficiency (Czech et al 2019b). This aspect becomes important when the catalytic efficiency and robustness of ectoine hydroxylases are benchmarked against each other when EctD enzymes are used in chemical biology approaches to hydroxylate substrates other than ectoine (Czech et al 2016(Czech et al , 2019bGalinski et al 2009;Hara et al 2019).…”

“…These enzymes seemed to possess similar kinetic parameters, but the subsequent optimization of assay conditions for individual enzymes revealed significant differences in their catalytic efficiency (Czech et al 2019b). This aspect becomes important when the catalytic efficiency and robustness of ectoine hydroxylases are benchmarked against each other when EctD enzymes are used in chemical biology approaches to hydroxylate substrates other than ectoine (Czech et al 2016(Czech et al , 2019bGalinski et al 2009;Hara et al 2019).…”

“…This metabolic profile, also referred to as underground metabolism, can be exploited by microbial cells to develop novel functions (D'Ari and Casadesus 1998;Jensen 1976). Recent studies have already exploited the substrate promiscuity of the EctD enzyme towards ectoine-related substrates; e. g., aiming at the selective hydroxylation of Lproline and of the synthetic ectoine derivative homoectoine (Czech et al 2019b;Galinski et al 2009;Hara et al 2019). Hence, ectoine hydroxylases have already found interesting uses in chemical biology.…”