Ectoine hydroxylase displays selective trans-3-hydroxylation activity towards l-proline

Hara, Ryoichi; Nishikawa, Takeyuki; Okuhara, Takuya; Koketsu, Kento; Kino, Kuniki

doi:10.1007/s00253-019-09868-y

Cited by 9 publications

(14 citation statements)

References 34 publications

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“…It endows the newly formed 5-hydroxyectoine (Inbar and Lapidot, 1988) with novel stress-protective and function preserving properties (Pastor et al, 2010; Czech et al, 2018a). Hence, the idea arose to exploit possible biosynthetic side activities of the ectoine hydroxylase as a catalyst in synthetic chemistry (Galinski et al, 2009; Hara et al, 2019). An attractive starting molecule for this approach is the synthetic ectoine derivative homoectoine (Schnoor et al, 2004) (Figure 1).…”

Section: Discussionmentioning

confidence: 99%

“…Hydroxylated prolines are interesting building blocks for medical and biotechnological applications as these can be incorporated in cyclic non-ribosomal peptide compounds, such as the antifungal agent echinocandin or the anti-tuberculosis drugs griselimycins (Houwaart et al, 2014; Lukat et al, 2017; Zhang et al, 2018). In a recent study, the EctD enzymes from H. elongata and Streptomyces cattleya were utilized to produce hydroxyprolines (Hara et al, 2019). While EctD from H. elongata only catalyzed the formation of trans -3-hydroxyproline from L -proline, the ( Sc )EctD enzyme also accepted 3,4-dehydro- L -proline, 2-methyl- L -proline, and L -pipecolic acid as substrates, highlighting that notable differences in the substrate profiles and kinetic parameters (Figures 4B–D) of bona fide ectoine hydroxylases exist (Höppner et al, 2014; Widderich et al, 2014a; Czech et al, 2018a).…”

Section: Discussionmentioning

confidence: 99%

“…All residues important for substrate/co-substrate binding and enzyme catalysis protrude into the lumen of the cupin barrel that also contains the catalytically critical Fe(II) atom (Höppner et al, 2014; Widderich et al, 2014b) (Figure 2B). Hence, the large barrel-like structure of the EctD monomer might be conducive to accommodating and accurately positioning compounds structurally and chemically related to ectoines and proline (Galinski et al, 2009, 2015; Hara et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2019

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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.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

et al. 2019

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“…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).…”

Section: The Ectoine Hydroxylase: Ectdmentioning

confidence: 99%

“…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.…”

Section: The Ectoine Hydroxylase: Ectdmentioning

confidence: 99%

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

Hermann

Mais

Czech

et al. 2020

Biological Chemistry

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Ectoine and its derivative 5-hydroxyectoine are compatible solutes and chemical chaperones widely synthesized by Bacteria and some Archaea as cytoprotectants during osmotic stress and high- or low-growth temperature extremes. The function-preserving attributes of ectoines led to numerous biotechnological and biomedical applications and fostered the development of an industrial scale production process. Synthesis of ectoines requires the expenditure of considerable energetic and biosynthetic resources. Hence, microorganisms have developed ways to exploit ectoines as nutrients when they are no longer needed as stress protectants. Here, we summarize our current knowledge on the phylogenomic distribution of ectoine producing and consuming microorganisms. We emphasize the structural enzymology of the pathways underlying ectoine biosynthesis and consumption, an understanding that has been achieved only recently. The synthesis and degradation pathways critically differ in the isomeric form of the key metabolite N-acetyldiaminobutyric acid (ADABA). γ-ADABA serves as preferred substrate for the ectoine synthase, while the α-ADABA isomer is produced by the ectoine hydrolase as an intermediate in catabolism. It can serve as internal inducer for the genetic control of ectoine catabolic genes via the GabR/MocR-type regulator EnuR. Our review highlights the importance of structural enzymology to inspire the mechanistic understanding of metabolic networks at the biological scale.

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Enzymatic reactions and microorganisms producing the various isomers of hydroxyproline

Kino

2020

Appl Microbiol Biotechnol

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Ectoine hydroxylase displays selective trans-3-hydroxylation activity towards l-proline

Cited by 9 publications

References 34 publications

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

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

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

Enzymatic reactions and microorganisms producing the various isomers of hydroxyproline

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