Photocontrolling the Enantioselectivity of a Phosphotriesterase via Incorporation of a Light-Responsive Unnatural Amino Acid

The external control of catalytic activity and substrate specificity of enzymes by light has aroused great interest in the fields of biocatalysis and pharmacology. Going beyond, we have attempted to photocontrol enzyme stereoselectivity on the example of phosphotriesterase (PTE), which is capable of hydrolyzing a wide variety of racemic organophosphorus substrates where one of two enantiomers is often highly toxic. To pursue this goal, the photocaged unnatural amino acid <i>o</i>-nitrobenzyl-l-tyrosine (ONBY) was incorporated by genetic code expansion at the large subsite of the active center, together with additional mutations at the small subsite. The stereoselectivities of the resulting PTE variants were tested with the achiral control substrate paraoxon and four different racemic substrates, which contained a <i>p</i>-nitrophenol leaving group in combination with either methyl-phenyl, ethyl-phenyl, methyl-cyclohexyl, or ethyl-cyclohexyl substituents. Comparison of the enantioselectivities (<i>k</i> _cat/<i>K</i> _M for S_p divided by <i>k</i> _cat/<i>K</i> _M for R_p) before and after decaging of ONBY using irradiation revealed the desired photoinduced inversion of enantioselectivity for three of the variants: PTE_I106A-H257ONBY exhibited a 43-fold stereoselectivity switch for the methyl-phenyl substrate, PTE_I106A-F132A-H257ONBY a 184-fold stereoselectivity switch for the methyl-cyclohexyl substrate, and PTE_I106A-F132A-S308A-H257ONBY a 52-fold and a 57-fold stereoselectivity switch for the methyl-cyclohexyl and the ethyl-cyclohexyl substrates. A computational analysis including molecular dynamics simulations and docking showed that a complicated interplay between steric constraints and specific enzyme-substrate interactions is responsible for the observed effects. Our findings significantly broaden the scope of possibilities for the spatiotemporal control of enantioselective transformations using light in biocatalytic systems.