Scheme 6. Cofactor Regeneration Systems in ene reductase catalysed hydrogenation reactions.
GDH = glucose dehydrogenase, G6PDH = glucose 6-phosphate dehydrogenase, FDH = formate dehydrogenase, ADH = alcohol dehydrogenase and PDH = phosphite dehydrogenase.[23]
facial selectivity are extremely rare and substrate specific among the OYE
fami-ly.[73,84,128,129] To get stereocomplementary products, a flipped binding mode has
to be enabled, for high enantiomeric excess even selectivity favoured over the normal binding mode. This, especially for bulkier substrates, could be difficult because of steric hindrance in the active site (see Figure 8).
2 O BJECTIVE OF T HE T HESIS
This thesis focuses on the engineering of ene reductases from the Old Yellow Enzyme (OYE) family. These new ene reductases will have superior substrate scope, stereocomplementarity and handling properties to establish them as in-dustrially relevant catalysts in organic synthesis. The work presented here is divided into four main parts:
Part One: Scaffold Sampling Strategy for the OYE Family
Analysing the existing knowledge about the OYE family from labor intensive directed evolution studies, will allow the identification of beneficial mutations to achieve desired properties, such as expanded substrate scope or stereocom-plementarity. The transferability of these engineered residues to other members of the OYE family to rationally design new catalysts is investigated here.
Figure 9. Working hypothesis for engineering the OYE family via the scaffold sampling strate-gy.
This so-called scaffold sampling strategy should reduce the search space in-volved in the directed evolution process, providing a shortcut to the discovery of new potent ene reductase variants, which should give access to both stereoi-somers of selected substrates.
Identified hot spot residues of YqjM from Bacillus subtilis for the model com-pound 3-methylcyclohex-2-en-1-one, i.e. C26D/I69T and C26G for activity and selectivity, respectively, will be transferred to seven OYE scaffolds. One of these scaffolds is a thermostable ER, which might be, due to its robustness, more in-teresting for applications in organic synthesis. This tested strategy would pro-vide a fast engineering method for this enzyme family allowing access to new, potent biocatalysts for organic synthesis.
Part Two: Characterization of a Robust and Stereocomplementary Panel of Ene Reductases from Thermus Scotoductus SA-01 (TsER)
The second section will cover the full characterization of a robust ene reductase variant panel from Thermus scotoductus SA-01 (TsER). Here, it will be demon-strated that TsER variant pairs can form a small panel of engineered ene reduc-tases that combine a broad substrate scope, tolerance to organic solvents and high temperature with convenient catalyst handling and control over facial se-lectivity (Figure 10). This combination of properties allows improved handling at gram-scale and conversion of poorly water-soluble compounds. The control over facial selectivity will be examined for a broad substrate scope.
In collaboration with D.J. OPPERMAN (University of the Free State, Bloemfon-tein), crystal structures of TsER variants will be obtained to provide structural insights into the factors controlling stereoselectivity and achieving activity to-wards non-substrates for the TsER wild type.
Figure 10. Characterization of ene reductase variants from Thermus scotoductus SA-01 (TsER) derived from part one. Investigation of properties and advantages of these variants for applica-tions in organic synthesis.
Part Three: Prediction of Substrate Binding and Affinity towards TsER variants by Computational Methods
As mentioned in the introduction a major challenge in the field of carbon-carbon reduction employing OYE homologues is the current lack of global un-derstanding of what governs selectivity and activity. To set up general rules the experimental studies will be examined with the help of fundamental computa-tional methods.
Theoretical studies, such as those employing docking, molecular dynamics simulations and hybrid quantum mechanics/molecular mechanics (QM/MM) simulations, can provide important insights into mechanistic details that may not be possible via experimental means. Relatively few theoretical studies have been performed on the OYE family.[130–132]
Performing in silico studies with the obtained TsER variant panel will gain a better understanding of the factors governing substrate acceptance and facial selectivity. More importantly, the docking data will be analysed to see whether the predicted stereochemistry matches the experimental observations.
Additionally, molecular dynamics simulations combined with methods for predicting binding free energies will clarify substrate affinity, which will allow a computational pre-screening for new substrate libraries.
Figure 11. Workflow for using computational methods to gain more information about the ex-perimental results.
Part Four: Development of a Set of Compounds as Molecular Probes for Active Site Geometry
In the fourth section, the substrate scope of TsER will be expanded to bulkier substrate classes such as indole and coumarin derivatives. The different couma-rin scaffolds, shown in Figure 12, should help to obtain insights into the binding pocket of TsER for the identification of potential new engineering residues. Due
to the fluorescent properties of the used coumarin derivatives, an easy and fast fluorescence screening will be established.
To achieve this, all coumarin scaffolds have first to be synthesised and second-ly, another round of mutagenesis must be performed to create a set of active site libraries. In general, there is a great interest in broadening the substrate scope of ene reductases to bulkier substrates to use these highly selective trans-hydrogenation catalysts in the late stage synthesis of complex organic mole-cules. So far the substrate scope is most often shown with small five and six membered ring compounds.
Figure 12. Different structural scaffolds based on coumarin (10a) to obtain insights into the ac-tive site of TsER for finding potential new engineering sides to convert more bulky substrates with ene reductases from the OYE family. Arrows are indicating possible, further introduced sites for hydrogen bonding.