Modulating regioselectivity of CYP107J3-catalyzed isophorone hydroxylation by disrupting the hydrophobic balance of the substrate binding pocket

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4-Hydroxyisophorone (4HIP) is an oxygenated intermediate derived from isophorone, serving as an important flavor and fragrance and chiral synthon of pharmaceutical drugs. In this study, a newly identified P450CYP107J3 from Bacillus cereus was found to prefer 4-hydroxylation of isophorone (80% regioselectivity) with 4HIP as the predominant product (59% product proportion). Bioinformatic analyses, including homologous modelling and molecular docking, reveal that four potential “hotspot” residues (L98, T300, M401 and V296) located on both sides of the substrate in the active pocket may control regioselectivity. By introducing polar residues to these hotspots to disrupt the hydrophobic balance, significant improvement in C4-regioselectivity was achieved by variants of L98, T300, and M401, with the most effective L98N exhibiting a notable enhancement of 93% C4-regioselectivity including 81% of 4HIP. Following, the double mutant L98N-M401F further improved both C4-regioselectivity (97%) and the proportion of 4HIP (86%). Remarkably, variant V296T essentially shifted regioselectivity from 4HIP to 6-hydroxyisophorone (6HIP) with an 80% preference for the latter, indicating the crucial role of V296 in controlling regioselectivity. Subsequently, the mechanism of regioselectivity of isophorone hydroxylation catalyzed by CYP107J3 was revealed by computational analysis. Furthermore, we demonstrated the generality of conserved hotspots L98 and V296 in mediating regioselectivity control in the CYP107J family members like CYP107J1 and CYP107J5. Overall, our study not only expands the biocatalytic toolbox for producing 4HIP and the α-hydroxy ketone 6HIP but also provides efficient engineering strategy and knowledge for the regioselectivity control of P450s in potential applications.