The halogenase toolbox is growing: Re-programming and optimization of a L-proline cis-4-hydroxylase for the cis-3-halogenation of its native substrate
Over 5000 halogenated natural products are described and around 25% of small molecular drugs are halogenated. Thus, strategies are needed to incorporate halogens in a selective and sustainable way.
In their recent article “Re-programming and optimization of a L-proline cis-4-hydroxylase for the cis-3-halogenation of its native substrate” just accepted by ChemCatChem Athena Papadopoulou, Jasmin Meierhofer, Fabian Meyer, Takahiro Hayashi, Samuel Schneider, Emine Sager, Rebecca Maria Ursula Buller describe new enzymes for enzymatic radical halogenation.
Starting from a Fe-/α-ketoglutarate-dependent hydroxylase the team used directed evolution to re-program the enzyme to a halogenase that is able to stereo-and regioselectivly halogenale the C3 position of L-proline. The approach started by a bioinformatics analysis of 20 literature-known Fe-/α-ketoglutarate-dependent hydroxylases and the cloning of the respective genes into E.coli. Five enzymes were expressed as soluble proteins. Variants with a D/A to G substation in the active site were tested for halogenation of their natural products. The D108G variant of L-proline cis-4-hydroxylase from Sinorhizobium meliloti halogenated L-proline selectively at the C3 position. During four rounds of directed evolution the team evolved a variant with a 12-fold increase in apparent kcat and a 98-fold increase of kcat/KM compared to the initial halogenation biocatalyst. SmP4H-7 represents the first example of an enzyme catalyzing stereo- and region-selective chlorination of L-proline at the C3 position described in the current literature.