[摘要] Bacillus megaterium P450 BM3 is a fatty acid hydroxylase with selectivity for long chain substrates (C12–C20). Binding or activity with substrates of chain length 12 has not been reported. Rational mutagenesis was used to re-design the enzyme to encourage binding of short chain fatty acids (C4–C10). We show that wild-type P450 BM3 has activity and weak affinity for substrates as short as butyrate (C4). However, turnover/binding of short chain substrates is dramatically increased by introducing a novel substrate carboxylate binding site close to the heme. Mutant L181K shows catalytic efficiency (k cat/K M) increased >13-fold with butyrate, while the L75T/L181K double mutant has k cat/K M increased >15-fold with hexanoate and binding (K d) improved >28-fold for butyrate. Removing the arginine 47/lysine 51 carboxylate binding motif at the mouth of the active site disfavours binding of all fatty acids, indicating its importance in the initial recognition of substrates.
