Rational re-design of the substrate binding site of flavocytochrome P450BM3

Bacillus megaterium P450 BM3 is a fatty acid hydroxylase with selectivity for long chain substrates (C-12-C-20). Binding or activity with substrates of chain length < C-12 has not been reported. Rational mutagenesis was used to redesign the enzyme to encourage binding of short chain fatty acids (C-4-C-10). We show that wild-type P450 BM3 has activity and weak affinity for substrates as short as butyrate (C-4). 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. (C) 2000 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.