Due to the high levels of enantioselectivity and slightly better initial activity observed with wild-type (wt) P. hybrida ACCO, we selected this nonheme Fe enzyme as the template for the further development of an efficient C(sp 3 )-H fluorinating biocatalyst (Fig. ). Guided by the crystal structure of ACCO and our molecular docking studies, we first performed site-saturation mutagenesis (SSM) and screening by targeting amino acid residues in proximity to the nonheme Fe centre. Surprisingly, only a single beneficial mutation I184A with modest activity improvement was found among residues in the closest sphere of Fe (Fig. , entry 2 and Table ). In light of the relatively small size of ACCO's native substrate 1-aminocyclopropane-1-carboxylic acid (see the SI for further details), we postulated that substrate tunnel engineering might be required for this nonheme enzyme to better accommodate bulky non-native substrates. We thus turned our attention to amino acid residues in the b-sheets of the substrate tunnel in protein engineering. In each round of engineering, we selected four tunnel residues for SSM. The best performing variant from the four SSM libraries was then selected as the basis for further optimisation. Through iterative saturation mutagenesis (ISM) and screening, five beneficial mutations distal to the Fe centre were identified in the tunnel-defining b-sheets. Among these, residue 158 was found to play a critical role in modulating the fluorination activity and enantioselectivity. While the incorporation of K158N into ACCO I184A resulted in 1.4-fold higher yield and similar enantioselectivity (Fig. , entry 3, (2.3 ± 0.1)% yield, 19:81 e.r.), the K158I variant of ACCO I184A furnished 2a with 3.2-fold higher yield and inverted enantiopreference (entry 4, (4.5 ± 0.2)% yield, 74:26 e.r.). The inverted stereochemistry observed with ACCO I184A K158I relative to that with ACCO I184A is surprising, as the a-carbon of K158 is 10.8 Å away from the Fe centre. These results demonstrated that remote residues such as 158 could influence not only the activity but also the enantioselectivity of ACCO in the current unnatural C-H fluorination.