ABSTRACT
The activity of four native FDHs and four engineered FDH variants on 93 low molecular weight arenes was used to generate FDH substrate activity profiles. These profiles provided insights into how substrate class, functional group substitution, electronic activation, and binding impact FDH activity and selectivity. The enzymes studied could halogenate a far greater range of substrates than previously recognized, but significant differences in their substrate specificity and selectivity were observed. Trends between the electronic activation of each site on a substrate and halogenation conversion at that site were established, and these data, combined with docking simulations, suggest that substrate binding can override electronic activation even on compounds differing appreciably from native substrates. These findings provide a useful framework for understanding and exploiting FDH reactivity for organic synthesis.
ABSTRACT
Directed evolution is a powerful tool for optimizing enzymes, and mutagenesis methods that improve enzyme library quality can significantly expedite the evolution process. Here, we report a simple method for targeted combinatorial codon mutagenesis (CCM). To demonstrate the utility of this method for protein engineering, CCM libraries were constructed for cytochrome P450BM3, pfu prolyl oligopeptidase, and the flavin-dependent halogenase RebH; 10-26 sites were targeted for codon mutagenesis in each of these enzymes, and libraries with a tunable average of 1-7 codon mutations per gene were generated. Each of these libraries provided improved enzymes for their respective transformations, which highlights the generality, simplicity, and tunability of CCM for targeted protein engineering.
