*2.2. Identification of Key Residues for the Enantioselectivity of OYE2y*

It was previously reported that the variant W116F of OYE1 from *S. pastorianus* partly reversed the enantioselectivity in the neral reduction [22]. However, the same substitution at site W117 corresponding to W116 in OYE1 even lowered the *e.e.* value from 38.13% (*R*) to 24.01% (*R*) when (*E*/*Z*)-citral was tested as substrate, indicating that the enantioselectivity switch for OYE1 and OYE2y seemed different. Furthermore, the NAD(P)H-dependent enoate reductase (OYE2p) from *S. cerevisiae* YJM1341 was newly discovered for asymmetric reduction of (*E*/*Z*)-citral to (*R*)-citronellal with the *e.e.* value of 88.8% (*R*), with four amino acid residues—G13, A59, I289, and H330—in OYE2p different from S13, S59, V289, and R330 in OYE2y (Figure 2). Considering the difference of enantioselectivity between OYE2p and OYE2y, it was expected that S13, S59, V289, and/or R330 might be critical for the enantioselectivity. Then, the single substitutions were conducted to evaluate this expectation. The catalytic performance of the variants S13G, S59A, and V289I was similar to that of OYE2y (Table 1). The substitution R330 to H significantly increased the (*R*)-enantioselectivity from 38.13% to 86.88% when (*E*/*Z*)-citral was used as substrate. Remarkably, the enantioselectivity was reversed from 32.66% (*S*) to 71.92% (*R*) when (*Z*)-citral was tested. Except for sequence alignment, the identification of key residues was conducted through the single mutation on the randomly-selected residues. Through multiple mutation attempts, the variant P76M was discovered to benefit the (*R*)-enantioselectivity of OYE2y in the citral reduction (Table 1). The substitution P76 to M increased the (*E*/*Z*) citral-derived *e.e.* value from 38.13% (*R*) to 57.60% (*R*), while the enantioselectivity in the reduction of (*Z*)-citral was lowered from 32.66% (*S*) to 4.76% (*S*). Thus, both R330 and P76 were chosen as the targets for subsequent site saturation mutagenesis.

**Figure 2.** Structure-related sequence alignment between OYE2y and its homologous OYEs. 1OYB: PDB code of OYE1 from *S. pastorianus*; OYE2p: NAD(P)H-dependent enoate reductase from *S. cerevisiae* YJM1341. The secondary structural elements of 1OYB (α-helices, β-strands, T-turns, and η-helices) were indicated above the aligned sequences. The numbering shown was from 1OYB. A red background highlights conserved residues. , the positions where the amino acid residues differed between OYE2y and OYE2p; , key residues for catalytic activity. The figure was produced using ESPript 3.0 [32].


<sup>a</sup> 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.

#### *2.3. Site-Saturation Mutagenesis of R330 in OYE2y*

All R330X variants of OYE2y (X = one of the other 19 amino acids) were successfully expressed in *E. coli*. After the cells were harvested by centrifugation and then disrupted by ultrasonication, each variant with *N*-terminal His tag was purified using affinity chromatography. As shown in Figure S1, all 19 variant proteins remained in the soluble fraction, revealing that these substitutions did not decrease the solubility. In comparison with the wild type OYE2y, the R330 variants of OYE2y fell into three categories: R330P without catalytic activity, R330Y with similar (*R*)-stereoselectivity to OYE2y, and the other 17 variants with improved (*R*)-stereoselectivity (Table 2). R330P did not retain the yellow color, suggesting that its substitution might deactivate the coenzyme binding. When (*E*/*Z*)-citral was tested as substrate, the variants except R330Y and R330P increased the (*R*)-stereoselectivity but decreased the product yield to some extent. In contrast to the reduction of (*E*)-citral, those 17 variants showed more significant improvement of (*R*)-enantioselectivity in the reduction of (*Z*)-citral. Among them, the variants R330H, R330D, and R330W had superior catalytic performance in terms of activity and enantioselectivity.


**Table 2.** The catalytic performance of OYE2y and its R330X variants a.

<sup>a</sup> X represents any of 20 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.
