Steroidal C7β alcohols and their respective esters have shown significant promise as neuroprotective and anti‐inflammatory agents to treat chronic neuronal damage like stroke, brain trauma, and cerebral ischemia. Since C7 is spatially far away from any functional groups that could direct C−H activation, these transformations are not readily accessible using modern synthetic organic techniques. Reported here are P450‐BM3 mutants that catalyze the oxidative hydroxylation of six different steroids with pronounced C7 regioselectivities and β stereoselectivities, as well as high activities. These challenging transformations were achieved by a focused mutagenesis strategy and application of a novel technology for protein library construction based on DNA assembly and USER (Uracil‐Specific Excision Reagent) cloning. Upscaling reactions enabled the purification of the respective steroidal alcohols in moderate to excellent yields. The high‐resolution X‐ray structure and molecular dynamics simulations of the best mutant unveil the origin of regio‐ and stereoselectivity.

Engineering artificial enzymes with high activity and catalytic mechanism different from naturally occurring enzymes is a challenge in protein design. For example, many attempts have been made to obtain active hydrolases by introducing a Ser → Cys exchange at the respective catalytic triads, but this generally induced a breakdown of activity. We now report that this long-standing dogma no longer pertains, provided additional mutations are introduced by directed evolution. By employing Candida antarctica lipase B (CALB) as the model enzyme with the Ser-His-Asp catalytic triad, a highly active cysteine-lipase having a Cys-His-Asp catalytic triad and additional mutations W104V/A281Y/A282Y/V149G can be evolved, showing a 40-fold higher catalytic efficiency than wild-type CALB in the hydrolysis of 4-nitrophenyl benzoate, and tolerating bulky substrates. Crystal structures, kinetics, MD simulations and QM/MM calculations reveal dynamic features and explain all results, including the preference of a two-step mechanism involving the zwitterionic pair Cys105⁻/His224⁺ rather than a concerted process.