To guide the selection of key active-site residues for SSM and screening, we built models of the key benzaldehyde-derived Breslow intermediate housed in the enzyme's active using PyMOL. Residues within 4.0 Å of the benzaldehyde-derived Breslow intermediate were targeted for SSM. In each SSM library, 86 clones were selected and screened. For PpBFD H281A, among the SSM libraries we examined, a new single mutant PpBFD H281V was found to catalyze this asymmetric alkylation with significantly improved activity and slightly better enantioselectivity (75% yield, 98.5:1.5 e.r., Table , entry 2). To improve the activity of wt PfBAL, we first compared the structures and sequences of PfBAL and PpBFD to gain insights into the origin of their reactivity differences. Sequence alignment analysis of PfBAL and PpBFD showed that T481 of PfBAL aligns with L461 of PpBFD (see the SI for details). Residue 481 of PfBAL is located in the a-helix proximal to the thiazolium core of the TPP cofactor, indicating its importance in modulating enzyme activity. We thus evaluated the PfBAL T481X SSM library, leading to an improved variant PfBAL T481L affording 3a in 65% yield with inverted enantiopreference (entry 4). Thus, the same leucine residue at the 461/481 site of PpBFD and PfBAL displayed general activating effects in these two enzymes. Finally, PfBAL T481L A480L afforded further improved enantioselectivity although with lower activity (47% yield, 94.5:5.5 e.r., entry 5). Interestingly, the inclusion of this A480L mutation also resulted in the reversal of BAL's enantiopreference. With optimized photobiocatalytic conditions and a set of engineered TPP enzyme variants in hand, we set out to survey the substrate scope of this photobiocatalytic radical alkylation (Table ). Using PpBFD H281V, benzaldehyde substrates possessing an electron-withdrawing substituent at the paraposition, such as a fluorine (4b), a chlorine (4c), a bromine (4d), and a trifluoromethyl (4e) could be effectively transformed, providing the corresponding enantioenriched ketones in good yield and excellent enantioselectivity. Benzaldehyde derivatives with a para-electron-donating groups including a methyl (4f), a methoxy (4g) and a dimethylamino (4h) were also compatible. Using PpBFD H281V, meta-substituted benzaldehydes furnished excellent enantioselectivity but lower activity (4i). Substantially better yield of 4i was observed when PfBAL T481L was employed, indicating the complementary activity profiles of PpBFD H281V and PfBAL T481L. Additionally, a heterocyclic substrate 3pyridinecarboxaldehyde 2j also underwent this biotransformation to afford the radical coupling product 4j in 48% yield (72.5:27.5 e.r.) and 78% yield (95.5:4.5 e.r.) with PpBFD H281V and PfBAL T481L A480L, respectively. An ortho-fluorine substituent (4k) was also compatible. As a limitation of our current TPP enzyme variants, benzaldehydes with a large ortho-substituent such as a methyl and a methoxy could not be efficiently converted. Nevertheless, we note that in a previously established biocatalytic benzoin condensation, this limitation could be overcome through protein engineering of PpBFD. (rac)-2a We also examined the substrate scope with respect to the redox-active esters. It was found that N-hydroxyphthalimide esters bearing a para-fluorine (4l), para-chlorine (4m), and paramethyl (4n) group underwent this transformation with excellent yields and enantioselectivities. Thienyl-containing redox-active ester also represented an excellent substrate (4o). In addition, Nhydroxyphthalimide esters with an extended aliphatic chain including those bearing an ethyl (4p) and an n-propyl (4q) group were also viable substrates. Compared with T481L, PpBFD H281V displayed substantially higher activities for these substrates, showcasing its potential to engage a larger range of alkyl radical precursors. Furthermore, nonbenzylic secondary alkyl radicals could also be converted, leading to aliphatic abranched ketones (4r). Importantly, using PpBFD H281V as the biocatalyst, a-keto acid substrates could be readily transformed under our photobiocatalytic alkylation conditions, giving rise to optically active products with excellent enantioselectivity and similar activities as the corresponding benzaldehydes (Table (A)). In contrast to PpBFD H281V, PfBAL variants PfBAL T481L and PfBAL T481L A480L provided low levels of activity (6% yield, 28.5:71.5 e.r. and 0% yield, respectively). PpBFD H281V allowed a range of a-keto acids (Table 4(B)), including 4-fluoro (4b), 4methyl (4f), and 4-methoxy (4g) phenylglyoxylic acids, to be transformed with excellent efficiency and enantioselectivity. Substituted redox-active esters including 4-fluoro (4l), 4-chloro (4m) and 4-methyl (4n) as well as a-ethyl (4p) and propyl (4q) substrates were also compatible using PpBFD H281V as the biocatalyst. These results further demonstrated the distinct synthetic utility of unnatural TPP radical enzymes from the BFD subfamily in the transformation of a-keto acid substrates . To gain insights into the mechanism of this asymmetric photobiocatalytic alkylation process, we carried out the standard reaction using PpBFD H281V and PfBAL T481L in the presence of 3 equiv. of 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO, Scheme 2(A)). In the presence of TEMPO, formation of product 4a was not observed. Radical trapping products 6a and 7a were observed with PpBFD H281V and PfBAL T481L, suggesting the involvement of radical intermediates in this process. The instability of TEMPO-trapping product 6a precluded the quantitative analysis of this product. Furthermore, we studied the enantiomeric-ratio evolution with regard to both the radical coupling product 3a and the recovered redox-active ester 2a as a function of reaction time. With PpBFD H281V, the e.r. of the product (3a) remained constant (97.5:2.5 e.r.) throughout this process. Furthermore, no kinetic resolution of the racemic redoxactive ester 2a was observed, confirming the enantioconvergent nature of this free radical process. Using PfBAL T481L A480L as the biocatalyst, similar to PpBFD H281V, the enantiomeric ratio of 4a also remained constant (94.5:5.5 e.r.) and no resolution of 2a was observed. Slower conversions were observed with PfBAL T481L A480L relative to that with PpBFD H281V. When phenylglyoxylic acid 5a was used, using PpBFD H281V, the reaction profile was found to be similar to that with benzaldehyde 1a, further demonstrating the kinetic competency of BFD enzymes in the transformation involving a-keto acids. No 4a formation was observed when PfBAL T481L A480L was used as the biocatalyst.