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Designed Enzyme Catalyzed Reaction Pathways – Green Chemistry and Synthetic Biology

• Understanding of structure/function relationships in enzymes catalyzing hydrolysis of epoxides and oxidation of vicinal diols.
• Generation and isolation of enzyme biocatalysts for specific and efficient catalysis of reactions generating chiral and pro-chiral molecules useful as synthetic precursors.
• Construction of enzymes reliably performing complete organic multi-step syntheses involving reactants and intermediates with chiral centers.
• Robust in vivo production of designed fine chemicals afforded by stepwise enzyme catalyzed synthesis.

Enzyme catalysts are characterized by an unprecedented specificity for reactant molecules that will be acted upon. Often only a specific stereoisomer of a chiral molecule will be efficiently converted by a particular enzyme. Such specificity is of high value if applied to synthesis of organic compounds where product purity is essential. The use of enzymes in organic synthesis is increasing and has been reported in reactions involving diverse chemistry [1-3]. With genetic methodology facilitating directed evolution of protein structure, the scope of useful biocatalysts is likely to expand in the coming years, as will the range of synthesis protocols applying enzyme-based biocatalysts in production of fine chemicals and pharmaceuticals.

Applying enzymes as catalysts allows for aqueous solvents to be used and reactions run at moderate temperatures. This ensures both an environmentally safe line of production as well as a minimized hazard of reaction handling. Use of enzymes thereby contributes to sustainable approaches in the production of useful chemicals.

Chemical Reactions: Our research aims to provide new synthetic routes to modularly constructed chemical substances containing functional groups amenable to further functionalization by additional enzyme-catalyzed reactions or by traditional organic synthesis. By applying enzymes acting in sequence on a set of structurally related substrate compounds, catalyzing specific reaction types, spectra of product molecules of controlled diversity is generated. Structurally engineered enzymes are utilized for regio- and stereospecific production of hydroxycarbonyl compounds.

Chiral epoxides are converted to vicinal diols in reactions catalyzed by epoxide hydrolases. The diols are subsequently oxidized by dehydrogenases to form aldehydes or ketones. The applied enzymes are chosen according to catalytic function, substrate specificity, including stereospecificity as well as practical concerns e.g. stability, catalytic efficiency etc.. Fig. 1 illustrates an example synthesis of chiral hydroxycarbonyl compounds starting from chiral epoxides. The formed products also contains prochiral centers which may, upon subsequent derivatizations, be transformed into further arrays of chiral products.

Figure 1. Outline of stepwise synthesis by enzyme-afforded catalysis. Stereospecific hydrolytic ring opening of substituted epoxides catalyzed by epoxide hydrolase generates vicinal diols amenable to oxidation by diol dehydrogenases. The possible range of hydroxy carbonyl products shown in the outline may readily be further extended by derivatizations in either enzyme-catalyzed reaction steps or by organic synthesis.

Enzyme Catalysts: To produce a diversity of enzyme catalysts of differing substrate specificities we perform genetic engineering of existing enzymes. Properties such as high catalytic effiencies and stereoselectivity are selected for during the directed evolution. We are at present working on engineering a plant epoxide hydrolase, StEH1, and a diol dehydrogenase from E. coli, FucO. Both enzymes are potentially highly stereospecific in their actions and therefore expected to produce products of enriched enantiomeric excess [4-6].

Epoxide hydrolases StEH1 has shown to be a highly suitable enzyme catalyst. The enzyme is stereospecific, easy to produce, structurally sturdy after mutagenesis and valuable structure/function data have been gathered [4, 9-13]. We study the enzymology of StEH1 in combination with production of new epoxide hydrolase-based catalysts by directed evolution to...

• establish structure/function relationships of enzyme-catalyzed epoxide ring opening reactions to guide design and generation of new stereospecific biocatalysts.
• isolate new specific biocatalysts with desired functional and physicochemical properties.
• diversify the catalytic repertoire of StEH1 by introduction of synthetic auxiliary active-site groups in combination with directed evolution.

A diol dehydrogenase from Escherichia coli, propanediol oxidoreductase (FucO), oxidizes (S)-1,2-propane diol into the corresponding aldehyde. This enzyme belongs to the iron-dependent class III group of alcohol dehydrogenases [6]. The tertiary structure of the enzyme reveals an accessible substrate binding site adapted for binding of low-molecular weight diols such as its natural substrate (Fig. 2). The structure, however, also invites to manipulation through directed evolution to generate enzyme forms which may accommodate also larger substrates such as aromatic diols, as exemplified in Fig. 1.

Figure 2. Active site of FucO. Catalysis depends on an iron (II) ion (orange) tightly bound through coordination with protein side-chains. The entry form the dinucleotide cofactor (yellow) is from the left in the picture, whereas the diol substrate (blue) enters through the channel from the right. A narrow “waist” in this channel prevents the entry of larger diols into the active site. By random mutagenesis of indicated residues (red) lining the active-site entry, the channel will be widened to allow for binding and oxidation of also larger diols, such as phenylethyl and phenylpropyl diols formed from epoxide hydrolase reactions. (The image was created from the atomic coordinates in 1RRM).

Our studies on FucO aims to...

• establish structure/function relationships of enzyme-catalyzed oxidation of vicinal diols to guide design and generation of new stereospecific dehydrogenases.
• isolate new FucO enzyme variants, by directed evolution, with substrate specificities which can be employed to act on diol derivatives produced from StEH1-catalyzed hydrolysis of corresponding epoxides.

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