*2.4. Site-Saturation Mutagenesis of P76 in OYE2y*

Similar to R330X variants, all P76X variants of OYE2y were successfully expressed in *E. coli* and purified (Figure S2). However, the number of the P76 variants in the category without catalytic activity (P76Y, P76Q, P76D, P76E, P76R, P76H, P76F, P76W, and P76K) was obviously greater than that of R330 variants, suggesting that P76 could be also critical for the activity. The category with significantly improved enantioselectivity included P76C, P76S, P76M, P76G, and P76N, whereas the other five variants (P76A, P76V, P76T, P76L, and P76I) showed similar catalytic performance to that of OYE2y (Table 3). Similar to the trend in the R330X variants, higher (*R*)-stereoselectivity of OYE2y variants was accompanied by lower product yields. When (*E*/*Z*)-citral was used as substrate, the substitution of P76 to C increased the *e.e.* value from 44.13% (*R*) to 69.92% (*R*), but the yield was lowered from 89.51% to 49.65%. Particularly, the *e.e.* value in the reduction of (*Z*)-citral was partly reversed from 32.66% (*S*) to 37.50% (*R*).


**Table 3.** The catalytic performance of OYE2y and its P76X variants a.

<sup>a</sup> X represents one of the other 19 amino acids. Data present mean values ± SD from three independent experiments. (*E*)-citral contained 98.38% geranial and 1.62% neral, (*Z*)-citral contained 96.84% neral and 3.16% geranial, and (*E*/*Z*)-citral contained 58.45% geranial and 41.55% neral. <sup>b</sup> "/" represents no catalytic activity.
