Cytochrome P450 BM3 from Bacillus megaterium catalyzes the hydroxylation of long-chain fatty acids and has no measurable activity on propane. This enzyme was converted into a highly efficient and specific propane monooxygenase over 13 rounds of directed evolution, , . The large change in substrate specificity was achieved using an incremental approach that involved screening first on an intermediate substrate. Since the native substrate contains a long alkane chain, and the target function was activity on a short alkane, an intermediate-length alkane towards which the parent enzyme had low but measurable activity (octane) was chosen as the initial directed evolution target. Once high octane activity was achieved, the selective pressure was switched toward activity on propane. a | Selected kinetic and biophysical properties of evolutionary intermediates from later generations. Total catalytic turnovers (moles propanol produced per mole P450), Km, and kcat are reported for propane hydroxylation. Thermostability is reported as T50 (temperature where half of the enzyme inactivates after 10 min incubation). Variants were selected for total propane activity in all generations, except for generation 9, which was selected for stability (T50). The mutations acquired during each generation are listed (and mapped to the structure below). Even small numbers of mutations can be responsible for large functional changes. Modified, with permission, from b | The crystal structure of the fifth generation P450 heme domain (139-3, PDB ID: 3CBD) with the locations of the mutations from subsequent generations colored as: generation 6 – red, generation 8 – green, generation 9 – blue, generation 10 – yellow, generation 11 – magenta, generation 12 – cyan, and generation 13 – orange. Beneficial mutations are distributed over the heme domain, and many are tens of Å from the catalytic iron.