Data archive of "Photo-Controlling Substrate Stereoselectivity of Phosphotriesterase via the Incorporation of a Light Responsive Non Canonical Amino Acid"

Organophosphates are a class of organophosphorus compounds that are used for a large variety of many different applications ranging from their use as insecticides, herbicides, or warfare agents to prodrugs used in pharmacology. First isolated from Pseudomonas diminuta and Flavobacterium sp. phosphotriesterase (PTE) has been shown to be capable of degrading various organophosphate compounds with the insecticide paraoxon as outstanding substrate where the rate of hydrolysis comes close to the rate limited by diffusion only. PTE features three distinct subsites for substrate binding, the small, the large and the leaving group subsite. Due to thedifferent sizes of these sites, PTE exhibits an enantioselectivity for the different stereoisomers (SP/RP) of chiral substrates. By altering the sizes of the subsites, stereoselectivity can be increased, relaxed, or inversed. Since the distinct enantiomers of a substrate may reveal significantly different biochemical properties, fine-tuning of enantioselectivity is highly desired to selectively degrade or isolate one single enantiomer. An interesting but hitherto unexplored way to tailor stereoselectivity is its light-control by photo-sensitive non-canonical amino acids (ncAAs). Based on this idea, in this work genetic code expansion was employed to create a photo-responsive PTE variant that exhibits an inversion of stereoselectivity upon light-exposure. More specifically, the photocaged ncAA o-nitrobenzyl-L-tyrosine (ONBY) was incorporated at two distinct positions the large subsite of PTE and further residue exchanges in both the small and the large subsite were selected based on rational design. In total, nine PTE variants containing ONBY were heterologously expressed and purified in sufficient amount and purity for further characterization. The enzymatic efficiencies and enantioselectivities of all variants for a defined substrate scope were determined by steady-state enzyme kinetics using a spectrophotometric assay. The preferentially hydrolyzed enantiomer by each variant was identified with a complementary assay employing the highly SP-enantiomer favoring PTE_G60A mutant and 31P NMR spectroscopy. The majority of the PTE variants did not display any significant effects indicative of an inversion of enantioselectivity upon irradiation for the racemic substrates tested. However, a slightly reversed stereoselectivity was observed for the hydrolysis of the cyclohexane-substituted substrates IV and V catalyzed by the variant PTE_I106A_F132A_H254G_H257ONBY, as well as for the hydrolysis of the phenyl-substituted substrate II catalyzed by PTE_H254G_L303T_H257ONBY. Strikingly, two other variants unambiguously exhibited a reversal of stereoselectivity upon irradiation. PTE_I106A_H257ONBY favored the SP enantiomer of substrate II by 3.5-fold before ONBY decaging while afterwards, the R¬P enantiomer was preferentially hydrolyzed by 36-fold. The photo-induced inversion of stereoselectivity was similar for PTE_I106A_F132A_S308A_H257ONBY where the preference of the SP enantiomer of substrate IV by 9-fold switched to a preference of the RP enantiomer by 18-fold. The design of a PTE variant whose stereoselectivity is inverted upon irradiation for the first time provides a fundamental starting point with regard to further investigations towards the photo-control of stereoselective enzymes and furthermore, offers advanced opportunities in various biochemical applications.